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NORMAL GENOMIC

• Huang LH, Lee WC, You ST, Cheng CC, Yu CJ
• Arfaptin-1 negatively regulates Arl1-mediated retrograde transport.
• PLoS One. 2015; 10: 118743-118743
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• The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1b (arfaptin-1b) as an Arl1-interacting protein and characterized a novel function for arfaptin-1 (including the arfaptin-1a and 1b isoforms) in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

• Rocca DL, Hanley JG
• PICK1 links AMPA receptor stimulation to Cdc42.
• Neurosci Lett. 2015; 585: 155-9
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• Rho-family GTPases control numerous cell biological processes via effects on actin dynamics, such as cell migration, cell adhesion, morphogenesis and vesicle traffic. In neurons, they are involved in dendritic spine morphogenesis and other aspects of neuronal morphology via regulation of the actin cytoskeleton. The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex. Excitatory synaptic transmission is known to regulate actin dynamics in dendritic spines to bring about changes in spine morphology or motility, however, the details of the signalling pathways that transduce glutamate receptor activation to Rho GTPase function are unclear. PICK1 is a PDZ and BAR domain protein that interacts with the Arp2/3 complex and the GTPase Arf1 to regulate actin polymerisation in dendritic spines. PICK1 also binds AMPA receptor subunits GluA2/3 and is involved in GluA2-dependent AMPAR trafficking. Here, we show that PICK1 binds Rac1 and Cdc42, via distinct but overlapping binding sites. Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process. This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.

• Nordmann D, Lickfeld M, Warnsmann V, Wiechert J, Jendretzki A, Schmitz HP
• The small GTP-binding proteins AgRho2 and AgRho5 regulate tip-branching, maintenance of the growth axis and actin-ring-integrity in the filamentous fungus Ashbya gossypii.
• PLoS One. 2014; 9: 106236-106236
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• GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.

• Rubashkin MG, Ou G, Weaver VM
• Deconstructing signaling in three dimensions.
• Biochemistry. 2014; 53: 2078-90
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• Cells in vivo exist within the context of a multicellular tissue, where their behavior is governed by homo- and heterotypic cell-cell interactions, the material properties of the extracellular matrix, and the distribution of various soluble and physical factors. Most methods currently used to study and manipulate cellular behavior in vitro, however, sacrifice physiological relevance for experimental expediency. The fallacy of such approaches has been highlighted by the recent development and application of three-dimensional culture models to cell biology, which has revealed striking phenotypic differences in cell survival, migration, and differentiation in genetically identical cells simply by varying culture conditions. These perplexing findings beg the question of what constitutes a three-dimensional culture and why cells behave so differently in two- and three-dimensional culture formats. In the following review, we dissect the fundamental differences between two- and three-dimensional culture conditions. We begin by establishing a basic definition of what "three dimensions" means at different biological scales and discuss how dimensionality influences cell signaling across different length scales. We identify which three-dimensional features most potently influence intracellular signaling and distinguish between conserved biological principles that are maintained across culture conditions and cellular behaviors that are sensitive to microenvironmental context. Finally, we highlight state-of-the-art molecular tools amenable to the study of signaling in three dimensions under conditions that facilitate deconstruction of signaling in a more physiologically relevant manner.

• Miertzschke M, Koerner C, Spoerner M, Wittinghofer A
• Structural insights into the small G-protein Arl13B and implications for Joubert syndrome.
• Biochem J. 2014; 457: 301-11
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• Ciliopathies are human diseases arising from defects in primary or motile cilia. The small G-protein Arl13B (ADP-ribosylation factor-like 13B) localizes to microtubule doublets of the ciliary axoneme and is mutated in Joubert syndrome. Its GDP/GTP mechanistic cycle and the effect of its mutations in patients with Joubert syndrome remain elusive. In the present study we applied high resolution structural and biochemical approaches to study Arl13B. The crystal structure of Chlamydomonas rheinhardtii Arl13B, comprising the G-domain and part of its unique C-terminus, revealed an incomplete active site, and together with biochemical data the present study accounts for the absence of intrinsic GTP hydrolysis by this protein. The structure shows that the residues representing patient mutations R79Q and R200C are involved in stabilizing important intramolecular interactions. Our studies suggest that Arg79 is crucial for the GDP/GTP conformational change by stabilizing the large two-residue register shift typical for Arf (ADP-ribosylation factor) and Arl subfamily proteins. A corresponding mutation in Arl3 induces considerable defects in effector and GAP (GTPase-activating protein) binding, suggesting a loss of Arl13B function in patients with Joubert syndrome.

• Liu Y, Kahn RA, Prestegard JH
• Interaction of Fapp1 with Arf1 and PI4P at a membrane surface: an example of coincidence detection.
• Structure. 2014; 22: 421-30
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• Interactions among ADP-ribosylation factors (ARFs), various adaptor proteins, and membrane lipids are essential for intracellular vesicle transport of a variety of cellular materials. Here, we present nuclear magnetic resonance (NMR)-based information on the nature of the interaction of yeast Arf1 (yArf1) and the pleckstrin homology (PH) domain of four-phosphate-adaptor protein 1 (Fapp1) as it occurs at a model membrane surface. Interactions favor a model in which Fapp1 is partially embedded in the membrane and interacts with a membrane-associated Arf1 molecule primarily through contacts between residues in switch I of Arf1 and regions near and under the solution exposed C-terminal extension of the PH domain. The Arf1 binding site on Fapp1-PH is distinct from a positively charged phosphatidylinositol-4-phosphate (PI4P) binding site. A structural model is constructed that supports coincidence detection of both activated ARF and PI4P as a mechanism facilitating Fapp1 recruitment to membranes.

• Holst B et al.
• PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.
• PLoS Biol. 2013; 11: 1001542-1001542
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• Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.

• Troeger A, Williams DA
• Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders.
• Exp Cell Res. 2013; 319: 2375-83
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• The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.

• Ismail SA, Chen YX, Miertzschke M, Vetter IR, Koerner C, Wittinghofer A
• Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119.
• EMBO J. 2012; 31: 4085-94
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• Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEdelta. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3.GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEdelta, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.

• Safari F, Suetsugu S
• The BAR Domain Superfamily Proteins from Subcellular Structures to Human Diseases.
• Membranes (Basel). 2012; 2: 91-117
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• Eukaryotic cells have complicated membrane systems. The outermost plasma membrane contains various substructures, such as invaginations and protrusions, which are involved in endocytosis and cell migration. Moreover, the intracellular membrane compartments, such as autophagosomes and endosomes, are essential for cellular viability. The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily proteins are important players in membrane remodeling through their structurally determined membrane binding surfaces. A variety of BAR domain superfamily proteins exist, and each family member appears to be involved in the formation of certain subcellular structures or intracellular membrane compartments. Most of the BAR domain superfamily proteins contain SH3 domains, which bind to the membrane scission molecule, dynamin, as well as the actin regulatory WASP/WAVE proteins and several signal transduction molecules, providing possible links between the membrane and the cytoskeleton or other machineries. In this review, we summarize the current information about each BAR superfamily protein with an SH3 domain(s). The involvement of BAR domain superfamily proteins in various diseases is also discussed.

• Mim C, Unger VM
• Membrane curvature and its generation by BAR proteins.
• Trends Biochem Sci. 2012; 37: 526-33
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• Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the Bin/amphiphysin/Rvs (BAR) domain proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR domain proteins sense, stabilize, and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent.

• Gehart H et al.
• The BAR domain protein Arfaptin-1 controls secretory granule biogenesis at the trans-Golgi network.
• Dev Cell. 2012; 23: 756-68
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• BAR domains can prevent membrane fission through their ability to shield necks of budding vesicles from fission-inducing factors. However, the physiological role of this inhibitory function and its regulation is unknown. Here we identify a checkpoint involving the BAR-domain-containing protein Arfaptin-1 that controls biogenesis of secretory granules at the trans-Golgi network (TGN). We demonstrate that protein kinase D (PKD) phosphorylates Arfaptin-1 at serine 132, which disrupts the ability of Arfaptin-1 to inhibit the activity of ADP ribosylation factor, an important component of the vesicle scission machinery. The physiological significance of this regulatory mechanism is evidenced by loss of glucose-stimulated insulin secretion due to granule scission defects in pancreatic beta cells expressing nonphosphorylatable Arfaptin-1. Accordingly, depletion of Arfaptin-1 leads to the generation of small nonfunctional secretory granules. Hence, PKD-mediated Arfaptin-1 phosphorylation is necessary to ensure biogenesis of functional transport carriers at the TGN in regulated secretion.

• Bonner SM et al.
• Sequence variation in promoter of Ica1 gene, which encodes protein implicated in type 1 diabetes, causes transcription factor autoimmune regulator (AIRE) to increase its binding and down-regulate expression.
• J Biol Chem. 2012; 287: 17882-93
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• ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC(+)) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity.

• Man Z, Kondo Y, Koga H, Umino H, Nakayama K, Shin HW
• Arfaptins are localized to the trans-Golgi by interaction with Arl1, but not Arfs.
• J Biol Chem. 2011; 286: 11569-78
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• Arfaptins (arfaptin-1 and arfaptin-2/POR1) were originally identified as binding partners of the Arf small GTPases. Both proteins contain a BAR (Bin/Amphiphysin/Rvs) domain, which participates in membrane deformation. Here we show that arfaptins associate with trans-Golgi membranes. Unexpectedly, Arl1 (Arf-like 1), but not Arfs, determines the trans-Golgi association of arfaptins. We also demonstrate that arfaptins interact with Arl1 through their BAR domain-containing region and compete for Arl1 binding with golgin-97 and golgin-245/p230, both of which also bind to Arl1 through their GRIP (golgin-97/RanBP2/Imh1p/p230) domains. However, arfaptins and these golgins show only limited colocalization at the trans-Golgi. Time-lapse imaging of cells overexpressing fluorescent protein-tagged arfaptins and golgin-97 reveals that arfaptins, but not golgin-97, are included in vesicular and tubular structures emanating from the Golgi region. These observations indicate that arfaptins are recruited onto trans-Golgi membranes by interacting with Arl1, and capable of inducing membrane deformation via their BAR domains.

• Kabaso D et al.
• Attachment of rod-like (BAR) proteins and membrane shape.
• Mini Rev Med Chem. 2011; 11: 272-82
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• Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain.

• Meierhofer T, Eberhardt M, Spoerner M
• Conformational states of ADP ribosylation factor 1 complexed with different guanosine triphosphates as studied by 31P NMR spectroscopy.
• Biochemistry. 2011; 50: 6316-27
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• Guanine nucleotide binding proteins (GNB-proteins) play an essential role in cellular signaling, acting as molecular switches, cycling between the inactive, GDP-bound form and the active, GTP-bound form. It has been shown that conformational equilibria also exist within the active form of GNB-proteins between conformational states with different functional properties. Here we present (31)P NMR data on ADP ribosylation factor 1 (Arf1), a GNB-protein involved in Golgi traffic, promoting the coating of secretory vesicles. To investigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety of commonly used GTP analogues, namely, GppCH(2)p, GppNHp, and GTPgammaS, were analyzed. To gain deeper insight into the conformational state of active Arf1, we titrated with Cu(2+)-cyclen and GdmCl and formed the complex with the Sec7 domain of nucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, from (31)P NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This state seems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effector domain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward a conformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchange factors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effector interacting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuning mechanism of regulation and thereby an interesting target for the modulation of protein activity.

• Wong VW et al.
• The autoimmunity-related GIMAP5 GTPase is a lysosome-associated protein.
• Self Nonself. 2010; 1: 259-268
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• A mutation in the rat GIMAP5 gene predisposes for autoimmunity, most famously in the BB rat model of autoimmune type 1 diabetes mellitus. This mutation is associated with severe peripheral T lymphopenia, as is mutation of the same gene in mice, but the mechanism by which GIMAP5 normally protects T cells from death is unknown. GIMAP5 is a putative small GTPase, a class of proteins which often fulfil their functions in the vicinity of cellular membranes. The objective of this study was to determine the normal intracellular location of GIMAP5 in lymphoid cells. Combining studies in rat, mouse and human systems, novel monoclonal antibodies (mAbs) were used to examine the localization of GIMAP5 and the closely-related protein, GIMAP1, in lymphoid cells by means of confocal microscopy and sub-cellular fractionation combined with immunoblotting. Additionally, human Jurkat T cells that inducibly express epitope-tagged GIMAP5 were established and used in electron microscopy (EM). Endogenous GIMAP5 was found to be located in a membraneous compartment/s which was also detected by established markers of lysosomes. GIMAP1, by contrast, was found to be located in the Golgi apparatus. EM studies of the inducible Jurkat T cells also found GIMAP5 in lysosomes and, in addition, in multivesicular bodies. This study establishes that the endogenous location of GIMAP5 is in lysosomes and related compartments and provides a clearer context for hypotheses about its mechanism of action.

• Lee SH, Dominguez R
• Regulation of actin cytoskeleton dynamics in cells.
• Mol Cells. 2010; 29: 311-25
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• The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.

• Jung AG, Labarrera C, Jansen AM, Qvortrup K, Wild K, Kjaerulff O
• A mutational analysis of the endophilin-A N-BAR domain performed in living flies.
• PLoS One. 2010; 5: 9492-9492
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• BACKGROUND: Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature was functional, another mutation (P143A, DeltaLEN) designed to decrease the curvature was not. CONCLUSIONS/SIGNIFICANCE: Our results provide novel insight into the structure/function relationship of the endophilin-A BAR domain in vivo, especially with relation to synaptic function.

• Kwon E, Kim DY, Gross CA, Gross JD, Kim KK
• The crystal structure Escherichia coli Spy.
• Protein Sci. 2010; 19: 2252-9
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• Escherichia coli spheroplast protein y (EcSpy) is a small periplasmic protein that is homologous with CpxP, an inhibitor of the extracytoplasmic stress response. Stress conditions such as spheroplast formation induce the expression of Spy via the Cpx or the Bae two-component systems in E. coli, though the function of Spy is unknown. Here, we report the crystal structure of EcSpy, which reveals a long kinked hairpin-like structure of four alpha-helices that form an antiparallel dimer. The dimer contains a curved oval shape with a highly positively charged concave surface that may function as a ligand binding site. Sequence analysis reveals that Spy is highly conserved over the Enterobacteriaceae family. Notably, three conserved regions that contain identical residues and two LTxxQ motifs are placed at the horizontal end of the dimer structure, stabilizing the overall fold. CpxP also contains the conserved sequence motifs and has a predicted secondary structure similar to Spy, suggesting that Spy and CpxP likely share the same fold.

• McGrath MF, de Bold AJ
• Transcriptional analysis of the mammalian heart with special reference to its endocrine function.
• BMC Genomics. 2009; 10: 254-254
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• BACKGROUND: Pharmacological and gene ablation studies have demonstrated the crucial role of the endocrine function of the heart as mediated by the polypeptide hormones ANF and BNP in the maintenance of cardiovascular homeostasis. The importance of these studies lies on the fact that hypertension and chronic congestive heart failure are clinical entities that may be regarded as states of relative deficiency of ANF and BNP. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify gene transcripts that underlie the phenotypic differences associated with the endocrine function of the heart. RESULTS: Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between the atria and ventricles based on a 1.8 fold cut-off. The identification of numerous chamber specific transcripts, such as ANF for the atria and Irx4 for the ventricles among several others, support the soundness of the GeneChip data and demonstrates that the differences in gene expression profiles observed between the atrial and ventricular tissues were not spurious in nature. Pathway analysis revealed unique expression profiles in the atria for G protein signaling that included Galphao1, Ggamma2 and Ggamma3, AGS1, RGS2, and RGS6 and the related K+ channels GIRK1 and GIRK4. Transcripts involved in vesicle trafficking, hormone secretion as well as mechanosensors (e.g. the potassium channel TREK-1) were identified in relationship to the synthesis, storage and secretion of hormones. CONCLUSION: The data developed in this investigation describes for the first time data on gene expression particularly centred on the secretory function of the heart. This provides for a rational approach in the investigation of determinants of the endocrine of the heart in health and disease.

• Prinz WA, Hinshaw JE
• Membrane-bending proteins.
• Crit Rev Biochem Mol Biol. 2009; 44: 278-91
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• Cellular membranes can assume a number of highly dynamic shapes. Many cellular processes also require transient membrane deformations. Membrane shape is determined by the complex interactions of proteins and lipids. A number of families of proteins that directly bend membranes have been identified. Most associate transiently with membranes and deform them. These proteins work by one or more of three types of mechanisms. First, some bend membranes by inserting amphipathic domains into one of the leaflets of the bilayer; increasing the area of only one leaflet causes the membrane to bend. Second, some proteins form a rigid scaffold that deforms the underlying membrane or stabilizes an already bent membrane. Third, some proteins may deform membranes by clustering lipids or by affecting lipid ordering in membranes. Still other proteins may use novel but poorly understood mechanisms. In this review, we summarize what is known about how different families of proteins bend membranes.

• Worby CA et al.
• The fic domain: regulation of cell signaling by adenylylation.
• Mol Cell. 2009; 34: 93-103
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• We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, huntingtin yeast-interacting protein E (HYPE), also adenylylates Rho GTPases in vitro. Thus, we classify Fic domain-containing proteins as a class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic posttranslational modification that may regulate key signaling events.

• Cardenas J, Rivero S, Goud B, Bornens M, Rios RM
• Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms.
• BMC Biol. 2009; 7: 56-56
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• BACKGROUND: The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. RESULTS: By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. CONCLUSION: We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.

• Phillips MJ, Calero G, Chan B, Ramachandran S, Cerione RA
• Effector proteins exert an important influence on the signaling-active state of the small GTPase Cdc42.
• J Biol Chem. 2008; 283: 14153-64
• Display abstract

• GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTP-bound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl beta,gamma-methylene-diphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP- and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP- and GMP-PCP-bound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCP-bound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP- and GDP-bound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.

• Buffa L, Fuchs E, Pietropaolo M, Barr F, Solimena M
• ICA69 is a novel Rab2 effector regulating ER-Golgi trafficking in insulinoma cells.
• Eur J Cell Biol. 2008; 87: 197-209
• Display abstract

• Islet cell autoantigen of 69kDa (ICA69) is a small GTPase-binding protein of unknown function. ICA69 is enriched in the Golgi complex and its N-terminal half contains a BAR domain, a module that can bind/bend membranes and interacts with phospholipids. Here we show that in insulinoma INS-1 cells ICA69 binds to the small GTPase Rab2, which regulates the transport of COPI vesicles between the endoplasmic reticulum and the Golgi complex. Rab2 binds to ICA69 in a GTP-dependent fashion and recruits it to membranes. Over-expression of either Rab2 or ICA69 in INS-1 cells results in a phenotype characterized by: (i) impaired anterograde transport of the secretory granule protein precursors pro-ICA512 and chromogranin A; (ii) reduction of stimulated insulin secretion. Taken together, these data identify ICA69 as a novel Rab2 effector and point to its role in regulating the early transport of insulin secretory granule proteins.

• Ugolev Y, Berdichevsky Y, Weinbaum C, Pick E
• Dissociation of Rac1(GDP).RhoGDI complexes by the cooperative action of anionic liposomes containing phosphatidylinositol 3,4,5-trisphosphate, Rac guanine nucleotide exchange factor, and GTP.
• J Biol Chem. 2008; 283: 22257-71
• Display abstract

• Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac.RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac.RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219 ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of "resting cell membrane" composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP).RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P(3) in the liposomes. Dissociation of Rac1(GDP).RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P(3) and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P(3) responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP).RhoGDI complexes, p67(phox), GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P(3) at a level superior to that of native membranes.

• Cotton M, Boulay PL, Houndolo T, Vitale N, Pitcher JA, Claing A
• Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration.
• Mol Biol Cell. 2007; 18: 501-11
• Display abstract

• ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.

• Li X et al.
• Involvement of a Golgi-resident GPI-anchored protein in maintenance of the Golgi structure.
• Mol Biol Cell. 2007; 18: 1261-71
• Display abstract

• The Golgi apparatus consists of a series of flattened cisternal membranes that are aligned in parallel to form stacks. Cytosolic-oriented Golgi-associated proteins have been identified that may coordinate or maintain the Golgi architecture. Here, we describe a novel GPI-anchored protein, Golgi-resident GPI-anchored protein (GREG) that has a brefeldin A-sensitive Golgi localization. GREG resides in the Golgi lumen as a cis-oriented homodimer, due to strong interactions between coiled-coil regions in the C termini. Dimerization of GREG as well as its Golgi localization depends on a unique tandem repeat sequence within the coiled-coil region. RNA-mediated interference of GREG expression or expression of GREG mutants reveals an essential role for GREG in maintenance of the Golgi integrity. Under these conditions, secretion of the vesicular stomatitis virus glycoprotein protein as a marker for protein transport along the secretory pathway is inhibited, suggesting a loss of Golgi function as well. These results imply the involvement of a luminal protein in Golgi structure and function.

• Viaud J et al.
• Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.
• Proc Natl Acad Sci U S A. 2007; 104: 10370-5
• Display abstract

• Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.

• Luo R et al.
• Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.
• Biochem J. 2007; 402: 439-47
• Display abstract

• Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

• Shiba T et al.
• Structural basis for Rab11-dependent membrane recruitment of a family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1.
• Proc Natl Acad Sci U S A. 2006; 103: 15416-21
• Display abstract

• Family of Rab11-interacting protein (FIP)3/Arfophlin-1 and FIP4/Arfophilin-2 are dual effectors for Rab11 and ADP ribosylation factor (ARF)5/ARF6, which are involved in membrane delivery from recycling endosomes to the plasma membrane during cytokinesis. Here, we define the distinct C-terminal binding regions of FIP3 and FIP4 for Rab11 and ARF5/ARF6. Furthermore, we determined the crystal structure of Rab11 in complex with the Rab11-binding domain (RBD) of FIP3. The long amphiphilic alpha-helix of FIP3-RBD forms a parallel coiled-coil homodimer, with two symmetric interfaces with two Rab11 molecules. The hydrophobic side of the RBD helix is involved in homodimerization and mediates the interaction with the Rab11 switch 1 region, whereas the opposite hydrophilic side interacts with the Rab11 switch 2 and is the major factor contributing to the binding specificity. The bivalent interaction of FIP3 with Rab11 at the C terminus allows FIP3 to coordinately function with other binding partners, including ARFs.

• Jin M, Goldenring JR
• The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system.
• Biochim Biophys Acta. 2006; 1759: 281-95
• Display abstract

• Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

• Ren G, Vajjhala P, Lee JS, Winsor B, Munn AL
• The BAR domain proteins: molding membranes in fission, fusion, and phagy.
• Microbiol Mol Biol Rev. 2006; 70: 37-120
• Display abstract

• The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes.

• Wasmeier C, Burgos PV, Trudeau T, Davidson HW, Hutton JC
• An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin.
• Traffic. 2005; 6: 474-87
• Display abstract

• Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.

• Lin PJ, Williams WP, Luu Y, Molday RS, Orlowski J, Numata M
• Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7.
• J Cell Sci. 2005; 118: 1885-97
• Display abstract

• The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Delta184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.

• Millard TH et al.
• Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53.
• EMBO J. 2005; 24: 240-50
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• The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.

• Li J et al.
• Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.
• Biochemistry. 2005; 44: 4365-74
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• Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

• Albrecht DE, Froehner SC
• DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes.
• J Biol Chem. 2004; 279: 7014-23
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• Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.

• Ritter B, Blondeau F, Denisov AY, Gehring K, McPherson PS
• Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics.
• Biochem Soc Trans. 2004; 32: 769-73
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• Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain. Accounting for identified fragmented peptides allows for a quantitative assessment of protein complexes associated with CCVs, and the identification of many of the known components of post-fusion synaptic vesicles demonstrates that a main function for brain CCVs is to recycle synaptic vesicles. In addition, we have identified a number of novel proteins that participate in CCV formation and function at the trans-Golgi network and the plasma membrane. Characterization of two of these proteins, NECAP1 and NECAP2, has led to the identification of a new consensus motif that mediates protein interactions with the clathrin adaptor protein 2. These studies highlight the ability of proteomics to reveal new insights into the mechanisms and functional roles of subcellular compartments.

• Charych EI et al.
• A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain.
• J Biol Chem. 2004; 279: 38978-90
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• We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor (GABA(A)R) clusters than with alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.

• Wilcox A, Katsanakis KD, Bheda F, Pillay TS
• Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex.
• J Biol Chem. 2004; 279: 38881-8
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• The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.

• Ambasudhan R et al.
• Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein.
• Genomics. 2004; 83: 615-25
• Display abstract

• Elongation of very long chain fatty acids 4 (ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.

• Rybakin V, Stumpf M, Schulze A, Majoul IV, Noegel AA, Hasse A
• Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus.
• FEBS Lett. 2004; 573: 161-7
• Display abstract

• Coronins constitute an evolutionary conserved family of WD-repeat actin-binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle-like cytoplasmic structures as well as on the cis-Golgi. In the Golgi region, crn7 staining appears broader than that of the cis-Golgi markers Erd2p and beta-COP, still, the trans-Golgi network appears predominantly crn7-negative. Importantly, the membrane-associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

• Hidaka S, Konecke V, Osten L, Witzgall R
• PIGEA-14, a novel coiled-coil protein affecting the intracellular distribution of polycystin-2.
• J Biol Chem. 2004; 279: 35009-16
• Display abstract

• Employing a yeast two-hybrid screen with the COOH terminus of polycystin-2, one of the proteins mutated in patients with polycystic kidney disease, we were able to isolate a novel protein that we call PIGEA-14 (polycystin-2 interactor, Golgi- and endoplasmic reticulum-associated protein with a molecular mass of 14 kDa). Molecular modeling only predicts a coiled-coil motif, but no other functional domains, in PIGEA-14. In a subsequent two-hybrid screen using PIGEA-14 as a bait, we found GM130, a component of the cis-compartment of the Golgi apparatus. Co-expression of the PIGEA-14 and PKD2 cDNAs in LLC-PK(1) and HeLa cells resulted in a redistribution of PIGEA-14 and polycystin-2 to the trans-Golgi network, which suggests that PIGEA-14 plays an important role in regulating the intracellular location of polycystin-2 and possibly other intracellular proteins. Our results also indicate that the intracellular trafficking of polycystin-2 is regulated both at the level of the endo-plasmic reticulum and that of the trans-Golgi network.

• Mitin NY, Ramocki MB, Zullo AJ, Der CJ, Konieczny SF, Taparowsky EJ
• Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization.
• J Biol Chem. 2004; 279: 22353-61
• Display abstract

• The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras.

• Haeusler LC, Blumenstein L, Stege P, Dvorsky R, Ahmadian MR
• Comparative functional analysis of the Rac GTPases.
• FEBS Lett. 2003; 555: 556-60
• Display abstract

• Small GTPases of the Rho family including Rac, Rho and Cdc42 regulate different cellular processes like reorganization of the actin cytoskeleton by acting as molecular switches. The three distinct mammalian Rac proteins share very high sequence identity but how their specificity is achieved is hitherto unknown. Here we show that Rac1 and Rac3 are very closely related concerning their biochemical properties, such as effector interaction, nucleotide binding and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis support the idea that altered dynamics of Rac2 at the switch I region may be responsible for different biochemical properties. These results provide insight into the individual functionalities of the Rac isoforms.

• Zhu PP et al.
• Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin.
• J Biol Chem. 2003; 278: 49063-71
• Display abstract

• Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.

• Paris S, Longhi R, Santambrogio P, de Curtis I
• Leucine-zipper-mediated homo- and hetero-dimerization of GIT family p95-ARF GTPase-activating protein, PIX-, paxillin-interacting proteins 1 and 2.
• Biochem J. 2003; 372: 391-8
• Display abstract

• ADP-ribosylation factor GTPase-activating proteins (ARFGAPs) of the G-protein-coupled receptor kinase interactor 1/p95 paxillin kinase linker/p95-ARFGAP Pak-interacting exchange factor paxillin-binding protein (APP)-1 family are multidomain proteins, which interact functionally with both ARF and Rac GTPases. These proteins are involved in the dynamic reorganization of adhesion and the cytoskeleton during cell motility. Our previous work [Di Cesare, Paris, Albertinazzi, Dariozzi, Andersen, Mann, Longhi and de Curtis (2000) Nat. Cell Biol. 2, 521-530] has pointed out a role for p95-APP1 in the regulation of ARF6-mediated membrane recycling. These proteins include different domains, and are capable of interacting stably with proteins that are supposed to play a role in the regulation of actin dynamics and adhesion. They contain a coiled-coil region comprising a putative leucine zipper, predicted to be involved in dimerization. In the present study, we have investigated the possibility that these proteins form dimers. Our results show that p95-APP1 forms homodimers and may also form heterodimers with the other member of the family, p95 paxillin kinase linker/p95-APP2. Both homo- and heterodimerization are disrupted by mutation of two leucine residues in the coiled-coil region of p95-APP1. The N-terminal portion of p95-APP1, including the ARFGAP domain, three ankyrin repeats and the Pak-interacting exchange factor-binding region, are not required for dimerization. Evidence is presented for the existence of endogenous oligomeric complexes. The implication of dimerization/oligomerization in the functioning of these proteins is discussed.

• Brugnera E et al.
• Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex.
• Nat Cell Biol. 2002; 4: 574-82
• Display abstract

• Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

• Sutterlin C, Hsu P, Mallabiabarrena A, Malhotra V
• Fragmentation and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells.
• Cell. 2002; 109: 359-69
• Display abstract

• The pericentriolar Golgi stacks are fragmented and found dispersed in mitotic mammalian cells. Addition of an antibody to the Golgi-associated protein GRASP65 inhibited Golgi fragmentation by mitotic cytosol in permeabilized cells. Microinjecting this antibody or the C-terminal fragment of GRASP65, which contains the antibody binding site, into normal rat kidney cells prevented entry into mitosis. Under these conditions the cells had completed S phase but were not in the prophase stage of mitosis. Fragmentation of the Golgi apparatus by nocodazole or Brefeldin A treatment prior to or post microinjection of the anti-GRASP65 antibody alleviated the block in mitotic entry. Based on our findings, we suggest that the pericentriolar Golgi organization is a sensor for controlling entry into mitosis in mammalian cells.

• Udby L, Calafat J, Sorensen OE, Borregaard N, Kjeldsen L
• Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils.
• J Leukoc Biol. 2002; 72: 462-9
• Display abstract

• Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense.

• Austin C, Boehm M, Tooze SA
• Site-specific cross-linking reveals a differential direct interaction of class 1, 2, and 3 ADP-ribosylation factors with adaptor protein complexes 1 and 3.
• Biochemistry. 2002; 41: 4669-77
• Display abstract

• We have used a site-specific photo-cross-linking approach to identify direct interactions between clathrin adaptor protein (AP)1 complexes and small GTPases of the ADP-ribosylation factor (ARF) family and to explore the specificity of this interaction on immature secretory granule (ISG) membranes. ISG membranes are a well-characterized, highly enriched preparation of membranes that has previously been shown to have the membrane-associated factors for ARF1 recruitment that are not present on artificial liposomes. All three classes of ARF proteins could be recruited to ISG membranes, displaying differential requirements for GTPgammaS. We found that ARF1, ARF5, and ARF6 interacted directly with the beta1-adaptin subunit of AP-1 in the presence of GTPgammaS. Furthermore, we observed a direct interaction between the switch 1 region of ARF1 and the N-terminal trunk domains of gamma- and beta1-adaptin. In addition, both ARF1 and ARF6 but not ARF5 interacted directly with the beta3- and delta-adaptin subunits of AP-3. No interaction was observed between AP-2 and any of the ARF proteins. Our results delineate the specificity and provide evidence of a direct interaction between different ARF proteins and the AP complexes AP-1 and AP-3 on natural ISG membranes and show that residues in the switch 1 region of ARF proteins can selectively bind to the trunk domains of these complexes.

• Santy LC, Casanova JE
• GTPase signaling: bridging the GAP between ARF and Rho.
• Curr Biol. 2002; 12: 3602-3602
• Display abstract

• Membrane traffic and actin cytoskeleton dynamics are intimately linked, and GTPases of the Rho and ARF families may work together to regulate both. Recent studies have identified a family of GTPase activating proteins (GAPs) that contain both ARF-GAP and Rho-GAP domains, providing the first direct link between these two signaling pathways.

• Paris S, Za L, Sporchia B, de Curtis I
• Analysis of the subcellular distribution of avian p95-APP2, an ARF-GAP orthologous to mammalian paxillin kinase linker.
• Int J Biochem Cell Biol. 2002; 34: 826-37
• Display abstract

• We describe here the identification and characterization of avian p95-APP2, a multi-domain protein of a recently identified family of ADP-ribosylation factor (ARF)-GTPase-activating proteins (GAPs) including mammalian G protein-coupled receptor kinases (GRK)-interactor 1 (GIT1), paxillin kinase linker (PKL), and GIT2, as well as avian p95-APP1. The p95-APP2 is eluted from Rac-GTP-gamma-S, but not from Rac-GDP-beta-S columns. As other members of the family, p95-APP2 has binding regions for the focal adhesion protein paxillin, and for the Rac exchanging factor PIX. Sequence comparison indicates that p95-APP2 is the avian orthologue of mammalian PKL. Expression studies showed a largely diffuse distribution of the full length p95-APP2, without evident effects on cell morphology. We observed a dramatic difference between the localization of the amino-terminal portion of the protein, including the ARF-GAP domain and the three ankyrin repeats, and the carboxy-terminal portion including the paxillin-binding site. Moreover, the expression of truncated carboxy-terminal polypeptides including both the PIX- and paxillin-binding regions leads to a marked localization of the protein together with paxillin at large vesicles. Comparison of the expression of corresponding ARF-GAP-deficient constructs from p95-APP2 and p95-APP1 shows their distribution at distinct endocytic compartments. Altogether, these data support a role of distinct members of this family of ARF-GAPs in the regulation of different steps of membrane traffic during cell motility, and suggest that p95-APP2 may shuttle between an intracellular compartment and the cell periphery, although, further work will be needed to address this point.

• Puertollano R, Randazzo PA, Presley JF, Hartnell LM, Bonifacino JS
• The GGAs promote ARF-dependent recruitment of clathrin to the TGN.
• Cell. 2001; 105: 93-102
• Display abstract

• The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

• Freyberg Z, Sweeney D, Siddhanta A, Bourgoin S, Frohman M, Shields D
• Intracellular localization of phospholipase D1 in mammalian cells.
• Mol Biol Cell. 2001; 12: 943-55
• Display abstract

• Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

• Wang JW, Howson J, Haller E, Kerr WG
• Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins.
• J Immunol. 2001; 166: 4586-95
• Display abstract

• Mutations in chs1/beige result in a deficiency in intracellular transport of vesicles that leads to a generalized immunodeficiency in mice and humans. The function of NK cells, CTL, and granulocytes is impaired by these mutations, indicating that polarized trafficking of vesicles is controlled by CHS1/beige proteins. However, a molecular explanation for this defect has not been identified. Here we describe a novel gene with orthologues in mice, humans, and flies that contains key features of both chs1/beige and A kinase anchor genes. We designate this novel gene lba for LPS-responsive, beige-like anchor gene. Expression of lba is induced after LPS stimulation of B cells and macrophages. In addition, lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Strikingly, LBA-green-fluorescent protein (GFP) fusion proteins are localized to vesicles after LPS stimulation. Confocal microscopy indicates this protein is colocalized with the trans-Golgi complex and some lysosomes. Further analysis by immunoelectron microscopy demonstrates that LBA-GFP fusion protein can localize to endoplasmic reticulum, plasma membrane, and endocytosis vesicles in addition to the trans-Golgi complex and lysosomes. We hypothesize that LBA/CHS1/BG proteins function in polarized vesicle trafficking by guiding intracellular vesicles to activated receptor complexes and thus facilitate polarized secretion and/or membrane deposition of immune effector molecules.

• Cabezon E, Runswick MJ, Leslie AG, Walker JE
• The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase.
• EMBO J. 2001; 20: 6990-6
• Display abstract

• In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.

• Grizot S, Faure J, Fieschi F, Vignais PV, Dagher MC, Pebay-Peyroula E
• Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation.
• Biochemistry. 2001; 40: 10007-13
• Display abstract

• A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

• Sadacharan SK, Cavanagh AC, Gupta RS
• Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules.
• Histochem Cell Biol. 2001; 116: 507-17
• Display abstract

• Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

• Poussu AM, Thompson PH, Makinen MJ, Lehto VP
• Vear, a novel Golgi-associated protein, is preferentially expressed in type I cells in skeletal muscle.
• Muscle Nerve. 2001; 24: 127-9
• Display abstract

• Vear is a novel Golgi-associated protein with a domain structure characteristic of many vesicular transport-associated proteins. It has been suggested that Vear is involved in vesicle transport through trans-Golgi. In this study, we have determined the localization of Vear in skeletal muscle. The staining for Vear in normal human muscle revealed a distribution pattern similar to that of type I fibers. We conclude that Vear is preferentially expressed in type I fibers in human muscle, presumably indicative of a specific function that remains to be identified.

• Komine Y, Eggink LL, Park H, Hoober JK
• Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components.
• Planta. 2000; 210: 897-905
• Display abstract

• The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtii y1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins.

• Morgans C, Brandstatter JH
• SNAP-25 is present on the Golgi apparatus of retinal neurons.
• Neuroreport. 2000; 11: 85-8
• Display abstract

• SNAP-25 is a neuronal SNARE protein required for synaptic vesicle exocytosis and neurite outgrowth. Here we show that in addition to synaptic staining, SNAP-25 immunoreactivity is also localized to an intracellular, perinuclear compartment of retinal neurons. Double-labeling with an antibody against the 58 kD resident protein of the trans-golgi network indicates that the intracellular SNAP-25 is localized to the Golgi complex. Immuno-electron microscopic localization of SNAP-25 confirmed its presence on the Golgi apparatus of photoreceptors, bipolar cells, amacrine cells and ganglion cells in the retina. These data implicate SNAP-25 in the trafficking of Golgi-derived vesicles in neurons in addition to the synaptic vesicle cycle.

• Di Cesare A et al.
• p95-APP1 links membrane transport to Rac-mediated reorganization of actin.
• Nat Cell Biol. 2000; 2: 521-30
• Display abstract

• Motility requires protrusive activity at the cellular edge, where Rho family members regulate actin dynamics. Here we show that p95-APP1 (ArfGAP-putative, Pix-interacting, paxillin-interacting protein 1), a member of the GIT1/PKL family, is part of a complex that interacts with Rac. Wild-type and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ADP-ribosylation factor 6 (Arf6). Distinct p95-APP1-derived polypeptides have different distributions, indicating that p95-APP1 cycles between the cell surface and endosomes. Our results show that p95-APP1 functionally interacts with Rac and localizes to endosomal compartments, thus identifying p95-APP1 as a molecular link between actin organization, adhesion, and membrane transport during cell motility.

• Mizoguchi T et al.
• Determination of functional regions of p125, a novel mammalian Sec23p-interacting protein.
• Biochem Biophys Res Commun. 2000; 279: 144-9
• Display abstract

• The Sec23p-Sec24p complex is a component of coat protein II-coated vesicles involved in protein export from the endoplasmic reticulum. We previously identified a novel Sec23p-interacting protein, p125, which consists of 1000 amino acids and comprises a proline-rich region and a phospholipase A(1) homology region. p125, when ectopically expressed in cultured cells, localizes to endoplasmic reticulum-Golgi intermediate regions. In the present study we showed that expressed p125 principally colocalizes with p115 and GM130, both of which are involved in vesicle tethering to Golgi membranes. Next, we determined the functional regions of p125 by expressing a p125 series with deletions. The results showed that the proline-rich region (residues 135-259) is responsible for the binding to Sec23p. For the correct localization of p125, a region (residues 135-1000) comprising both the proline-rich and phospholipase A(1) homology regions was required.

• Scheffzek K, Stephan I, Jensen ON, Illenberger D, Gierschik P
• The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.
• Nat Struct Biol. 2000; 7: 122-6
• Display abstract

• Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.

• Poussu A, Lohi O, Lehto VP
• Vear, a novel Golgi-associated protein with VHS and gamma-adaptin "ear" domains.
• J Biol Chem. 2000; 275: 7176-83
• Display abstract

• The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to gamma-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker gamma-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane- and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.

• Pilon M, Peng XR, Spence AM, Plasterk RH, Dosch HM
• The diabetes autoantigen ICA69 and its Caenorhabditis elegans homologue, ric-19, are conserved regulators of neuroendocrine secretion.
• Mol Biol Cell. 2000; 11: 3277-88
• Display abstract

• ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting beta-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into the ric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.

• Kippenberger AG, Palmer DJ, Comer AM, Lipski J, Burton LD, Christie DL
• Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.
• J Neurochem. 1999; 73: 1024-32
• Display abstract

• The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

• Garrido PA et al.
• Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway.
• DNA Cell Biol. 1999; 18: 323-31
• Display abstract

• Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.

• Day GJ, Mosteller RD, Broek D
• Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner.
• Mol Cell Biol. 1998; 18: 7444-54
• Display abstract

• The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 [alpha3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.

• Record E et al.
• Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae.
• Can J Microbiol. 1998; 44: 945-53
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• The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy. The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes. A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium. The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus. All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.

• Hennig D, Scales SJ, Moreau A, Murley LL, De Mey J, Kreis TE
• A formiminotransferase cyclodeaminase isoform is localized to the Golgi complex and can mediate interaction of trans-Golgi network-derived vesicles with microtubules.
• J Biol Chem. 1998; 273: 19602-11
• Display abstract

• A protein of 60 kDa (p60) has been identified using a quantitative in vitro vesicle-microtubule binding assay. Purified p60 induces co-sedimentation with microtubules of trans-Golgi network-derived vesicles isolated from polarized, perforated Madin-Darby canine kidney cells. Sequencing of the cDNA coding for this protein revealed that it is the chicken homologue of formiminotransferase cyclodeaminase (FTCD), a liver-specific enzyme involved in the histidine degradation pathway. Purified p60 from chicken liver has formiminotransferase activity, confirming that it is FTCD or an isoform of this enzyme. Isoforms of FTCD were identified in chicken hepatoma and HeLa cells, and immunolocalize to the region of the Golgi complex and vesicular structures in its vicinity. Furthermore, 58K, a previously identified microtubule-binding Golgi protein from rat liver (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092), is identical to FTCD. Both proteins co-purify with microtubules and co-localize with membranes of the Golgi complex. The capacity of FTCD to bind both to microtubules and Golgi-derived membranes may suggest that this protein, or one of its isoforms, might have in addition to its enzymatic activity, a second physiological function in mediating interaction of Golgi-derived membranes with microtubules.

• Zen K, Notarfrancesco K, Oorschot V, Slot JW, Fisher AB, Shuman H
• Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane.
• Am J Physiol. 1998; 275: 17283-17283
• Display abstract

• Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic betaHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.

• Qu-Hong, Brown LF, Dvorak HF, Dvorak AM
• Ultrastructural immunogold localization of osteopontin in human gastric mucosa.
• J Histochem Cytochem. 1997; 45: 21-33
• Display abstract

• We performed ultrastructural immunogold localization of osteopontin in the mucosa of human stomach. This adhesive glycoprotein was present in mucous and chief cells of the epithelial layer and in macrophages in the lamina propria. Parietal and endocrine cells of the epithelial layer and mast cells and plasma cells in the lamina propria did not contain osteopontin, serving as internal negative controls. Subcellular localizations of osteopontin included secretory granules and synthetic organelles in mucous and chief cells and phagolysosomes in macrophages. Extracellular concentrations of osteopontin were present in the glycocalyx and in an electron-lucent band between epithelial surface cells and the gastric lumen. Paracellular edema between the epithelium of the same cells was devoid of osteopontin. Immunogold localization of pepsinogen II was done to identify cells with mixed granule populations and contents of multicompartmental secretory granules. These studies revealed mucous cell granules and chief cell granules, each containing compartmentalized storage products, which included osteopontin and mucigen in mucous cells and osteopontin and pepsinogen II in chief cells. Cytochemical controls for the immunogold localizations were negative. The subcellular distribution of osteopontin in human gastric mucosa suggests possible roles for this glycoprotein in barrier function, host defense, and/or secretion.

• Calafat J, Janssen H, Knol EF, Weller PF, Egesten A
• Ultrastructural localization of Charcot-Leyden crystal protein in human eosinophils and basophils.
• Eur J Haematol. 1997; 58: 56-66
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• The Charcot-Leyden crystal (CLC) protein with lysophospholipase activity and carbohydrate-binding properties is a characteristic constituent of eosinophils and basophils. We investigated its subcellular distribution using immunoelectron microscopy. Eosinophil progenitors, mature eosinophils and basophils all contained CLC protein in their cytosol and in the euchromatin of the nucleus. A minor population of granules in eosinophils, increasing in number with maturation, and a more abundant granule-population in basophils, were found to contain CLC protein. Double-labeling experiments showed, in eosinophils, that CLC protein-containing granules contain also eosinophil peroxidase, a characteristic specific granule protein. This suggests a relationship between the CLC protein-containing organelle and the specific granule. In basophils both the CLC protein positive and the negative granules showed the same characteristic particulate-like structure of the granular matrix and both share the same membrane marker CD63. In nasal polyps, macrophages were observed phagocytosing necrotic eosinophils. In these macrophages CLC protein-containing vesicles were observed, probably representing late endosomes. The dual (cytosolic/nuclear and granular) localization of CLC protein suggests that this protein enters both a secretory and a nonsecretory pathway during its biosynthesis, indicating functional roles for this protein both within the cell and extracellularly.

• Vitale N, Moss J, Vaughan M
• Characterization of a GDP dissociation inhibitory region of ADP-ribosylation factor domain protein ARD1.
• J Biol Chem. 1997; 272: 25077-82
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• ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and later recognized as critical components in intracellular vesicular transport and phospholipase D activation. ARF domain protein 1 (ARD1) is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We previously reported that this extension acts as a GTPase-activating protein for the ARF domain of ARD1 (Vitale, N., Moss, J., and Vaughan, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1941-1944). Both GTP binding and GTP hydrolysis are necessary for physiological function of guanine nucleotide-binding proteins, and the rates of GDP/GTP exchange and GTPase activity are critical in the activation/deactivation cycle. Dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (both proteins synthesized in Escherichia coli). Using deletion mutations, it was demonstrated that the 15 amino acids directly preceding the ARF domain were responsible for decreasing the rate of GDP dissociation but not guanosine 5-[gamma-thio]triphosphate dissociation. By site-specific mutagenesis it was shown that hydrophobic amino acids in this region were particularly important in stabilizing the GDP-bound form of ARD1. It is suggested that, like the amino-terminal segment of ARF, the equivalent region in ARD1, located between the GTPase-activating protein and ARF domains, may act as a GDP dissociation inhibitor.

• Cluett EB, Kuismanen E, Machamer CE
• Heterogeneous distribution of the unusual phospholipid semilysobisphosphatidic acid through the Golgi complex.
• Mol Biol Cell. 1997; 8: 2233-40
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• To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.

• Solimena M et al.
• ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules.
• EMBO J. 1996; 15: 2102-14
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• Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti-ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin-containing granules.

• Lim L, Manser E, Leung T, Hall C
• Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways.
• Eur J Biochem. 1996; 242: 171-85
• Display abstract

• The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42-->Rac-->Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.

• Mally MI, Cirulli V, Hayek A, Otonkoski T
• ICA69 is expressed equally in the human endocrine and exocrine pancreas.
• Diabetologia. 1996; 39: 474-80
• Display abstract

• Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.

• Funaki T, Fujiwara T, Hong HS, Misumi Y, Nishioka M, Ikehara Y
• Identification and characterization of a 230-kDa Golgi-associated protein recognized by autoantibodies from a patient with HBV hepatitis.
• Cell Struct Funct. 1996; 21: 63-72
• Display abstract

• A serum from a patient with HBV hepatitis was found to contain autoantibodies reacting with various mammalian cells. Immunofluorescence staining of cultured cells with the autoantibodies revealed that the antigen was localized at perinuclear regions, where the Golgi markers alpha-mannosidase II and beta-COP were colocalized. The autoantigen disappeared from the perinuclear regions upon incubation with the fungal metabolite brefeldin A, and the immunostainable structures were fragmented into vesicles by treatment with nocodazole. These results strongly indicate that the antigen is localized at the Golgi complex. Immunoblots of cell lysates showed that the autoantibodies recognized a single protein with a molecular mass of 230 kDa in a variety of cell lines, indicating that the 230-kDa antigen is a conserved protein among mammalian species. We designated this protein GCP230 (Golgi complex-associated protein with a molecular mass of 230 kDa). when a postnuclear fraction was prepared and centrifuged, GCP230 was recovered in both cytosol and membrane fractions. Peripheral interaction of GCP230 with membranes was confirmed by phase separation in Triton X-114 solution and by extraction with sodium carbonate. Taken together, these results indicate that GCP230 is a peripheral membrane protein of the Golgi derived from the cytosol, although its function is not known at present.

• Sengupta D, Gumkowski FD, Tang LH, Chilcote TJ, Jamieson JD
• Localization of cellubrevin to the Golgi complex in pancreatic acinar cells.
• Eur J Cell Biol. 1996; 70: 306-14
• Display abstract

• Cellubrevin is the smallest (14 kDa) isoform of the synaptobrevin (VAMP) protein family and is found in a wide variety of tissues. Western blot analysis with a polyclonal antibody against the unique N-terminus of cellubrevin identified a protein of 14 kDa in rat pancreas. This protein distributed predominantly to the particulate fractions from the rat exocrine pancreas and was totally resistant to NaHCO3 washes, indicating that it is an integral membrane protein. Subcellular fractionation of pancreatic homogenates showed enrichment of this protein in the smooth microsomal fraction while negligible amounts were present in the zymogen granule membrane or the rough microsomal membrane fractions. As seen in other tissues, the 14 kDa immunoreactive form was proteolyzed by tetanus toxin. Light and electron microscopic immunocytochemistry localized cellubrevin immunoreactivity primarily to small vesicles and condensing vacuoles originating from the Golgi region, with significantly lower labeling on zymogen granules. Based on the intracellular localization of cellubrevin detected in acinar cells by immunocytochemistry and cell fractionation, we suggest that cellubrevin may be involved in the maturation of secretory granules.

• Christopher GK, Sundermann CA
• Intracellular insulin binding in Tetrahymena pyriformis.
• Tissue Cell. 1996; 28: 427-37
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• Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the alpha-subunit of the human insulin receptor (amino acid residues 657-670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30-50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.

• Yamashina S, Tamaki H
• The significance of lamellated structure of Golgi apparatus in parotid acinar cells.
• Eur J Morphol. 1996; 34: 229-32
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• By reviewing briefly our recent study, the functional significance of lamellated structure of the Golgi apparatus (GA) was discussed. We have examined the structural alterations and distribution of Golgi related proteins in GA disorganized by brefeldin A (BFA), okadaic acid (OA), monensin and nocodazole (NOC) by means of electron microscopy. BFA and OA induced rapid disruption of lamellated structure of GA into groups of small vesicles and tubules. The effect of these drugs was reversible, and lamellated structure recovered immediately after withdrawal of the drugs. Immunoelectron microscopy revealed that the localization of amylase, GF-1 antigen (a resident protein of trans membrane of GA) and cathepsin D was modified considerably in the cells whose lamellated structures were in the middle of disorganization or reconstruction. These modifications include appearance of cathepsin D and GF-1 immunoreactivity in the secretory granules. Furthermore, there was evidence that secretory products were discharged not only into the acinar lumen but also into the baso-lateral spaces. In contrast, the stacked configuration was preserved in GA after treatment with monensin or NOC. Localization of immunoreactivity of the Golgi related proteins was essentially not changed by these drugs. These findings suggested that the cisternal stack of GA was the structural background for the normal sorting of secretory proteins.

• Schmitz F, Wallis KT, Rho M, Drenckhahn D, Murphy DB
• Intracellular distribution of kinesin in chromaffin cells.
• Eur J Cell Biol. 1994; 63: 77-83
• Display abstract

• In this paper we examined the association of the microtubule motor protein kinesin with organelles in chromaffin cells. Approximately 15% of kinesin was associated with membranes as determined by differential and equilibrium centrifugation on sucrose gradients. Kinesin was not enriched in a particular organelle fraction but cofractionated with a variety of organelle markers including markers for early and late endosomes, smooth and rough endoplasmic reticulum (ER) and the Golgi apparatus. Surprisingly, low amounts of kinesin were present in fractions of purified chromaffin granules. The absence of kinesin from the bulk of chromaffin granules was also indicated by immunostaining of tissue sections. A polyclonal antibody that specifically recognized the 120 kDa kinesin heavy chain labeled predominantly a perinuclear region that is typical for most of the kinesin-binding organelles identified by cell fractionation (endosomes, Golgi, ER). Since these organelles are compartments with high membrane turnover, we speculate that kinesin might be involved in certain aspects of trafficking of these membrane systems.

• Youakim A, Dubois DH, Shur BD
• Localization of the long form of beta-1,4-galactosyltransferase to the plasma membrane and Golgi complex of 3T3 and F9 cells by immunofluorescence confocal microscopy.
• Proc Natl Acad Sci U S A. 1994; 91: 10913-7
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• beta-1,4-Galactosyltransferase (GalTase) is localized to two subcellular compartments, the Golgi complex, where it participates in cellular glycosylation, and the plasma membrane, where it functions as a receptor for oligosaccharide ligands on opposing cells or in the extracellular matrix. The gene for GalTase encodes two nearly identical proteins that differ only in their N-terminal cytoplasmic domains: both short and long GalTases share an 11-aa cytoplasmic tail, but long GalTase has an additional 13-aa sequence on its cytoplasmic domain. In this study, we investigated the subcellular distribution of endogenous long GalTase in untransfected F9 and 3T3 cells by using confocal microscopy and antibodies specific for the 13-aa sequence unique to long GalTase. Long GalTase was found in the Golgi complex as expected; long GalTase was also found on the plasma membrane in cell-type-specific distributions. In 3T3 cells, long GalTase was evident on the basal surface of cells possessing a migratory phenotype, being concentrated at the leading and trailing edges; nonmigratory cells had little detectable surface immunoreactivity. In F9 cells, long GalTase was localized on the plasma membrane, being concentrated at the apical aspect of intercellular junctions. These results demonstrate that in 3T3 and F9 cells, long GalTase is present on the cell surface in addition to the Golgi complex. The pattern of surface expression shows cell-type specificity that is consistent with GalTase function in cellular interactions.

• Solimena M, Dirkx R Jr, Radzynski M, Mundigl O, De Camilli P
• A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region.
• J Cell Biol. 1994; 126: 331-41
• Display abstract

• The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.

• Amor JC, Harrison DH, Kahn RA, Ringe D
• Structure of the human ADP-ribosylation factor 1 complexed with GDP.
• Nature. 1994; 372: 704-8
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• ADP-ribosylation factors (ARFs) are essential and ubiquitous in eukaryotes, being involved in vesicular transport and functioning as an activator of phospholipase D (refs 1, 2) and cholera toxin. The functions of ARF proteins in membrane traffic and organelle integrity are intimately tied to its reversible association with membranes and specific interactions with membrane phospholipids. One common feature of these functions is their regulation by the binding and hydrolysis of GTP. Here we report the three-dimensional structure of full-length human ARF1 (M(r) 21,000) in its GDP-bound non-myristoylated form. The presence of a unique amino-terminal alpha-helix and loop, together with differences in Mg2+ ligation and the existence of a non-crystallographic dimer, set this structure apart from other GTP-binding proteins. These features provide a structural basis for the GTP-dependent modulation of membrane affinity, the lack of intrinsic GTPase activity, and the nature of effector binding surfaces.

• Sano K, Waguri S, Sato N, Kominami E, Uchiyama Y
• Coexistence of renin and cathepsin B in secretory granules of granular duct cells in male mouse submandibular gland.
• J Histochem Cytochem. 1993; 41: 433-8
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• Cathepsin B, a representative lysosomal cysteine proteinase, has been demonstrated to coexist with renin in secretary granules of rat pituitary LH/FSH cells and renal juxtaglomerular cells. We investigated immunocytochemically the localization of cathepsins B, H, and L in the submandibular gland of male mice, in which active renin is also produced. By light microscopy, granular immunodeposits for cathepsin B were detected in epithelial cells of the gland, particularly in granular duct cells and interstitial cells. Immunoreactivity for cathepsins H and L was mainly found in interstitial cells, although that for cathepsin H was weakly seen in acinar cells. By electron microscopy, immunogold particles indicating cathepsin B intensely labeled small granules near the Golgi complex of granular duct cells and weakly labeled large secretory granules, whereas those showing renin labeled both granules. Double immunostaining co-localized immunogold particles showing renin and cathepsin B in small perinuclear granules near the Golgi complex. Some immunopositive granules seemed to be closely associated with the Golgi elements. These results indicate that the co-localization of renin and cathepsin B is also seen in secretory granules of granular duct cells in the mouse submandibular gland, as seen in rat juxtaglomerular and LH/FSH cells. This suggests that cathepsin B is one of the possible candidates for the renin-processing enzyme.

• Guest PC, Ravazzola M, Davidson HW, Orci L, Hutton JC
• Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans.
• Endocrinology. 1991; 129: 734-40
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• The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.

• Brand SH, Laurie SM, Mixon MB, Castle JD
• Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.
• J Biol Chem. 1991; 266: 18949-57
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• A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.

• van Heumen WR, Roubos EW
• Ultrastructural evidence for synthesis, storage and release of insulin-related peptides in the central nervous system of Lymnaea stagnalis.
• Neuroscience. 1990; 39: 493-500
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• The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnalis, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets and (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.

• de Curtis I, Simons K
• Characterization of the trans-Golgi network in BHK cells.
• Prog Clin Biol Res. 1988; 270: 101-13

• Grimaldi KA, Hutton JC, Siddle K
• Production and characterization of monoclonal antibodies to insulin secretory granule membranes.
• Biochem J. 1987; 245: 557-66
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• Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.

• Saermark T, Jones PM, Robinson IC
• Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.
• Biochem J. 1984; 218: 591-9
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• We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis.

• Hutton JC, Peshavaria M, Tooke NE
• 5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.
• Biochem J. 1983; 210: 803-10
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• Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.

• LaBella FS, Vivian S, Bindler E
• Amino acid composition of neurohypophysial secretory granules and Van Dyke protein.
• Biochem Pharmacol. 1967; 16: 1126-30

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