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NORMAL GENOMIC

• Okamura M, Inose H, Masuda S
• RNA Export through the NPC in Eukaryotes.
• Genes (Basel). 2015; 6: 124-49
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• In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

• Asakawa H, Mori C, Ohtsuki C, Iwamoto M, Hiraoka Y, Haraguchi T
• Uncleavable Nup98-Nup96 is functional in the fission yeast Schizosaccharomyces pombe.
• FEBS Open Bio. 2015; 5: 508-14
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• Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98-Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98-Nup96 joint molecule as a sole protein product of the nup189 (+) gene in the fission yeast Schizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98-Nup96 is dispensable for cell growth, at least under normal culture conditions in S. pombe.

• Fogelgren B et al.
• Urothelial Defects from Targeted Inactivation of Exocyst Sec10 in Mice Cause Ureteropelvic Junction Obstructions.
• PLoS One. 2015; 10: 129346-129346
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• Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6-14 hours after birth. Sec10FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions.

• Asakawa H et al.
• Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe.
• Nucleus. 2014; 5: 149-62
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• The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8-47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC.

• Song Q, Johnson C, Wilson TE, Kumar A
• Pooled segregant sequencing reveals genetic determinants of yeast pseudohyphal growth.
• PLoS Genet. 2014; 10: 1004570-1004570
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• The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (Sigma1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In Sigma1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.

• Gerace E, Moazed D
• Coimmunoprecipitation of proteins from yeast.
• Methods Enzymol. 2014; 541: 13-26
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• This protocol outlines a procedure for testing whether two proteins interact. A target protein will be immunoprecipitated using an antibody that recognizes it (or a tagged version of the protein). The immunoprecipitated material will be separated by SDS-PAGE and analyzed by Western blotting to assess the presence of a candidate interacting protein(s).

• Zhang Y et al.
• DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells.
• Protein Cell. 2014; 5: 124-40
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• Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

• Russell JD et al.
• Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.
• PLoS One. 2013; 8: 58157-58157
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• Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the beta6 (PBF1) and beta7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

• Sugiyama T, Wanatabe N, Kitahata E, Tani T, Sugioka-Sugiyama R
• Red5 and three nuclear pore components are essential for efficient suppression of specific mRNAs during vegetative growth of fission yeast.
• Nucleic Acids Res. 2013; 41: 6674-86
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• Zinc-finger domains are found in many nucleic acid-binding proteins in both prokaryotes and eukaryotes. Proteins carrying zinc-finger domains have important roles in various nuclear transactions, including transcription, mRNA processing and mRNA export; however, for many individual zinc-finger proteins in eukaryotes, the exact function of the protein is not fully understood. Here, we report that Red5 is involved in efficient suppression of specific mRNAs during vegetative growth of Schizosaccharomyces pombe. Red5, which contains five C3H1-type zinc-finger domains, localizes to the nucleus where it forms discrete dots. A red5 point mutation, red5-2, results in the upregulation of specific meiotic mRNAs in vegetative mutant red5-2 cells; northern blot data indicated that these meiotic mRNAs in red5-2 cells have elongated poly(A) tails. RNA-fluorescence in situ hybridization results demonstrate that poly(A)(+) RNA species accumulate in the nucleolar regions of red5-deficient cells. Moreover, Red5 genetically interacts with several mRNA export factors. Unexpectedly, three components of the nuclear pore complex also suppress a specific set of meiotic mRNAs. These results indicate that Red5 function is important to meiotic mRNA degradation; they also suggest possible connections among selective mRNA decay, mRNA export and the nuclear pore complex in vegetative fission yeast.

• Reyes-Turcu FE, Zhang K, Zofall M, Chen E, Grewal SI
• Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin.
• Nat Struct Mol Biol. 2011; 18: 1132-8
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• Heterochromatin assembly at Schizosaccharomyces pombe centromeres involves a self-reinforcing loop mechanism wherein chromatin-bound RNAi factors facilitate targeting of Clr4-Rik1 methyltransferase. However, the initial nucleation of heterochromatin has remained elusive. We show that cells lacking Mlo3, a protein involved in mRNP biogenesis and RNA quality control, assemble functional heterochromatin in RNAi-deficient cells. Heterochromatin restoration is linked to RNA surveillance because loss of Mlo3-associated TRAMP also rescues heterochromatin defects of RNAi mutants. mlo3Delta, which causes accumulation of bidirectional repeat-transcripts, restores Rik1 enrichment at repeats and triggers de novo heterochromatin formation in the absence of RNAi. RNAi-independent heterochromatin nucleation occurs at selected euchromatic loci that show upregulation of antisense RNAs in mlo3Delta cells. We find that the exosome RNA degradation machinery acts parallel to RNAi to promote heterochromatin formation at centromeres. These results suggest that RNAi-independent mechanisms exploit transcription and non-coding RNAs to nucleate heterochromatin.

• Xu T et al.
• A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth.
• J Biol Chem. 2010; 285: 15476-88
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• Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1Delta mutants are noninvasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1Delta mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.

• Selvanathan SP, Thakurta AG, Dhakshnamoorthy J, Zhou M, Veenstra TD, Dhar R
• Schizosaccharomyces pombe Dss1p is a DNA damage checkpoint protein that recruits Rad24p, Cdc25p, and Rae1p to DNA double-strand breaks.
• J Biol Chem. 2010; 285: 14122-33
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• Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light Deltadss1 cells entered mitosis prematurely with indistinguishable timing from Deltarad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G(2)/M boundary in response to DNA damage.

• Yuan Q, Jantti J
• Functional analysis of phosphorylation on Saccharomyces cerevisiae syntaxin 1 homologues Sso1p and Sso2p.
• PLoS One. 2010; 5: 13323-13323
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• BACKGROUND: The Saccharomyces cerevisiae syntaxin1 homologues Sso1p and Sso2p perform an essential function in membrane fusion in exocytosis. While deletion of either SSO1 or SSO2 causes no obvious phenotype in vegetatively grown cells, deletion of both genes is lethal. In sporulating diploid S. cerevisiae cells only Sso1p, but not Sso2p, is needed for membrane fusion during prospore membrane formation. Mass spectrometry and in vivo labeling data suggest that serines 23, 24, and 79 in Sso1p and serines 31 and 34 in Sso2p can be phosphorylated in vivo. Here we set out to assess the contribution of phosphorylation on Sso protein in vivo function. PRINCIPAL FINDINGS: Different mutant versions of SSO1 and SSO2 were generated to target the phosphorylation sites in Sso1p and Sso2p. Basal or overexpression of phospho-mimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. However, S79A and S79E mutations caused a mild defect in the ability of Sso1p to complement the temperature-sensitive growth phenotype of sso2-1 sso1Delta cells. Combination of all mutations did not additionally compromise Sso1p in vivo function. When compared to the wild type SSO1 and SSO2, the phosphoamino acid mutants displayed similar genetic interactions with late acting sec mutants. Furthermore, diploid cells expressing only the mutant versions of Sso1p had no detectable sporulation defects. In addition to sporulation, also pseudohyphal and invasive growth modes are regulated by the availability of nutrients. In contrast to sporulating diploid cells, deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth. CONCLUSIONS: The identified phosphorylation sites do not significantly contribute to the in vivo functionality of Sso1p and Sso2p in S. cerevisiae.

• Zhang Y, Liu CM, Emons AM, Ketelaar T
• The plant exocyst.
• J Integr Plant Biol. 2010; 52: 138-46
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• The exocyst is an octameric vesicle tethering complex that functions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. All proteins in the complex have been conserved during evolution, and genes that encode the exocyst subunits are present in the genomes of all plants investigated to date. Although the plant exocyst has not been studied in great detail, it is likely that the basic function of the exocyst in vesicle tethering is conserved. Nevertheless, genomic and genetic studies suggest that the exocyst complex in plants may have more diversified roles than that in budding yeast. In this review, we compare the knowledge about the exocyst in plant cells to the well-studied exocyst in budding yeast, in order to explore similarities and differences in expression and function between these organisms, both of which have walled cells.

• Wheeler BS, Blau JA, Willard HF, Scott KC
• The impact of local genome sequence on defining heterochromatin domains.
• PLoS Genet. 2009; 5: 1000453-1000453
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• Characterizing how genomic sequence interacts with trans-acting regulatory factors to implement a program of gene expression in eukaryotic organisms is critical to understanding genome function. One means by which patterns of gene expression are achieved is through the differential packaging of DNA into distinct types of chromatin. While chromatin state exerts a major influence on gene expression, the extent to which cis-acting DNA sequences contribute to the specification of chromatin state remains incompletely understood. To address this, we have used a fission yeast sequence element (L5), known to be sufficient to nucleate heterochromatin, to establish de novo heterochromatin domains in the Schizosaccharomyces pombe genome. The resulting heterochromatin domains were queried for the presence of H3K9 di-methylation and Swi6p, both hallmarks of heterochromatin, and for levels of gene expression. We describe a major effect of genomic sequences in determining the size and extent of such de novo heterochromatin domains. Heterochromatin spreading is antagonized by the presence of genes, in a manner that can occur independent of strength of transcription. Increasing the dosage of Swi6p results in increased heterochromatin proximal to the L5 element, but does not result in an expansion of the heterochromatin domain, suggesting that in this context genomic effects are dominant over trans effects. Finally, we show that the ratio of Swi6p to H3K9 di-methylation is sequence-dependent and correlates with the extent of gene repression. Taken together, these data demonstrate that the sequence content of a genomic region plays a significant role in shaping its response to encroaching heterochromatin and suggest a role of DNA sequence in specifying chromatin state.

• Wilmes GM et al.
• A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing.
• Mol Cell. 2008; 32: 735-46
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• We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA-processing pathways. Due to their complexity and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here, we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.

• Gross T et al.
• The DEAD-box RNA helicase Dbp5 functions in translation termination.
• Science. 2007; 315: 646-9
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• In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.

• Tran EJ, Zhou Y, Corbett AH, Wente SR
• The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events.
• Mol Cell. 2007; 28: 850-9
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• Messenger RNA (mRNA) export involves the unidirectional passage of ribonucleoprotein particles (RNPs) through nuclear pore complexes (NPCs), presumably driven by the ATP-dependent activity of the DEAD-box protein Dbp5. Here we report that Dbp5 functions as an RNP remodeling protein to displace the RNA-binding protein Nab2 from RNA. Strikingly, the ADP-bound form of Dbp5 and not ATP hydrolysis is required for RNP remodeling. In vivo studies with nab2 and dbp5 mutants show that a Nab2-bound mRNP is a physiological Dbp5 target. We propose that Dbp5 functions as a nucleotide-dependent switch to control mRNA export efficiency and release the mRNP from the NPC.

• Cole CN, Scarcelli JJ
• Unravelling mRNA export.
• Nat Cell Biol. 2006; 8: 645-7

• Tuncher A, Sprote P, Gehrke A, Brakhage AA
• The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human.
• J Mol Biol. 2005; 352: 517-33
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• The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.

• Estruch F, Hodge CA, Rodriguez-Navarro S, Cole CN
• Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export.
• J Biol Chem. 2005; 280: 9691-7
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• Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.

• Fujita A, Hiroko T, Hiroko F, Oka C
• Enhancement of superficial pseudohyphal growth by overexpression of the SFG1 gene in yeast Saccharomyces cerevisiae.
• Gene. 2005; 363: 97-104
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• In response to nitrogen limitation, diploid yeast strains of Saccharomyces cerevisiae undergo a dimorphic transition to a filamentous growth form known as pseudohyphal growth. This developmental change can be classified into two distinct growing forms: invasive pseudohyphal growth and superficial pseudohyphal growth. We identified a yeast gene, SFG1, whose overexpression predominantly enhances superficial pseudohyphal growth when starved for nitrogen. Sfg1 has a sequence similarity to members of a family of transcriptional regulators of fungal development. Cells of a homozygous sfg1/sfg1 diploid strain have a serious defect in pseudohyphal growth, indicating that Sfg1 has an essential function for pseudohyphal development. Our analyses show that Sfg1 may act separately from mitogen-activated protein kinase (MAPK) pathway and cAMP-dependent protein kinase A (PKA) pathway.

• Knop M et al.
• Molecular interactions position Mso1p, a novel PTB domain homologue, in the interface of the exocyst complex and the exocytic SNARE machinery in yeast.
• Mol Biol Cell. 2005; 16: 4543-56
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• In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.

• Gilbert W, Guthrie C
• The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA.
• Mol Cell. 2004; 13: 201-12
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• mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.

• Demple B
• Grasping the message: regulated mRNA stability in free radical stress responses. Rodriguez-Gabriel MA, Burns G, McDonald WH et al. RNA-binding protein Csx1 mediates global control of gene expression in response to oxidative stress. EMBO J 2003; 22: 6256-6266.
• Redox Rep. 2004; 9: 3-5

• Vanoosthuyse V, Valsdottir R, Javerzat JP, Hardwick KG
• Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p.
• Mol Cell Biol. 2004; 24: 9786-801
• Display abstract

• Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

• Funakoshi M, Li X, Velichutina I, Hochstrasser M, Kobayashi H
• Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability.
• J Cell Sci. 2004; 117: 6447-54
• Display abstract

• Degradation of polyubiquitinated proteins by the proteasome often requires accessory factors; these include receptor proteins that bind both polyubiquitin chains and the regulatory particle of the proteasome. Overproduction of one such factor, Dsk2, is lethal in Saccharomyces cerevisiae and we show here that this lethality can be suppressed by mutations in SEM1, a gene previously recognized as an ortholog of the human gene encoding DSS1, which binds the BRCA2 DNA repair protein. Yeast sem1 mutants accumulate polyubiquitinated proteins, are defective for proteasome-mediated degradation and cannot grow under various stress conditions. Moreover, sem1 is synthetically lethal with mutations in proteasome subunits. We show that Sem1 is a component of the regulatory particle of the proteasome, specifically the lid subcomplex. Loss of Sem1 impairs the stability of the 26S proteasome and sem1Delta defects are greatly enhanced by simultaneous deletion of RPN10. The Rpn10 proteasome subunit appears to function with Sem1 in maintaining the association of the lid and base subcomplexes of the regulatory particle. Our data suggest a potential mechanism for this protein-protein stabilization and also suggest that an intact proteasomal regulatory particle is required for responses to DNA damage.

• Yoon JH
• Schizosaccharomyces pombe rsm1 genetically interacts with spmex67, which is involved in mRNA export.
• J Microbiol. 2004; 42: 32-6
• Display abstract

• We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMexl. The rsm1 gene contains no introns and encodes a 296 amino-acid-long protein with the RING finger domain, a C3HC4 in the N-terminal half. The deltarsm1 null mutant is viable, but it showed a slight poly(A)+ RNA accumulation in the nucleus. Also, the combination of deltarsm1 and deltaspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)+ RNA export defect. These results suggest that rsm1 is involved in mRNA export from the nucleus.

• Erkmann JA, Kutay U
• Nuclear export of mRNA: from the site of transcription to the cytoplasm.
• Exp Cell Res. 2004; 296: 12-20
• Display abstract

• Cellular mRNAs are produced in the nucleus and must be exported to the cytoplasm to allow for their translation into proteins. Recruitment of export factors to nascent mRNA starts cotranscriptionally and involves elaborate systems of quality control. Correctly processed mRNAs are committed for export in the form of large ribonucleoprotein complexes (mRNPs). Translocation of mRNPs through the nuclear pore complex (NPC) is mediated by a conserved heterodimeric transport receptor (NXF1/p15 in metazoa and Mex67p/Mtr2p in yeast) that bridges the interaction between the mRNP and the NPC. In this review, we describe the cis- and trans-requirements for mRNA export as well as the different mechanisms of recruiting export factors to mRNPs. We also discuss the significance of linking mRNA export with both downstream and upstream events in gene expression.

• Gould KL, Ren L, Feoktistova AS, Jennings JL, Link AJ
• Tandem affinity purification and identification of protein complex components.
• Methods. 2004; 33: 239-44
• Display abstract

• As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.

• Fujita M, Yoko-o T, Okamoto M, Jigami Y
• GPI7 involved in glycosylphosphatidylinositol biosynthesis is essential for yeast cell separation.
• J Biol Chem. 2004; 279: 51869-79
• Display abstract

• GPI7 is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. We isolated gpi7 mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature. WSC1, RHO2, ROM2, GFA1, and CDC5 genes were isolated as multicopy suppressors of gpi7-2 mutant. Multicopy suppressors could suppress the growth defect of gpi7 mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect of gpi7 mutants. Furthermore, deletion of EGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of the gpi7 mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating that GPI7 plays an important role in cell separation via the GPI-based modification of daughter-specific proteins in S. cerevisiae.

• Wang H, Tang X, Balasubramanian MK
• Rho3p regulates cell separation by modulating exocyst function in Schizosaccharomyces pombe.
• Genetics. 2003; 164: 1323-31
• Display abstract

• Cytokinesis is the final stage of the cell division cycle in which the mother cell is physically divided into two daughters. In recent years the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis, since it divides using an actomyosin ring whose constriction is coordinated with the centripetal deposition of new membranes and a division septum. The final step of cytokinesis in S. pombe requires the digestion of the primary septum to liberate two daughters. We have previously shown that the multiprotein exocyst complex is essential for this process. Here we report the isolation of rho3(+), encoding a Rho family GTPase, as a high-copy suppressor of an exocyst mutant, sec8-1. Overproduction of Rho3p also suppressed the temperature-sensitive growth phenotype observed in cells lacking Exo70p, another conserved component of the S. pombe exocyst complex. Cells deleted for rho3 arrest at higher growth temperatures with two or more nuclei and uncleaved division septa between pairs of nuclei. rho3Delta cells accumulate approximately 100-nm vesicle-like structures. These phenotypes are all similar to those observed in exocyst component mutants, consistent with a role for Rho3p in modulation of exocyst function. Taken together, our results suggest the possibility that S. pombe Rho3p regulates cell separation by modulation of exocyst function.

• Toikkanen JH, Miller KJ, Soderlund H, Jantti J, Keranen S
• The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae.
• J Biol Chem. 2003; 278: 20946-53
• Display abstract

• The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.

• Hwang CS, Oh JH, Huh WK, Yim HS, Kang SO
• Ssn6, an important factor of morphological conversion and virulence in Candida albicans.
• Mol Microbiol. 2003; 47: 1029-43
• Display abstract

• Candida albicans, the major fungal pathogen in humans, undergoes morphological conversion from yeasts to filamentous growth forms depending upon various environmental conditions. Here, we have identified a C. albicans gene, namely SSN6, encoding a putative global transcriptional co-repressor that is highly homologous to the Saccharomyces cerevisiae Ssn6. The isolated C. albicans SSN6 complemented the pleiotropic phenotypes of S. cerevisiae ssn6 mutation, and its expression levels declined significantly in response to a strong true hyphal inducer, serum. The mutant lacking C. albicans Ssn6 displayed a stubby pseudohyphal growth pattern, derepressed filament-specific genes in response to elevated temperature 37 degrees C and failed to develop true hyphae, extensive filamentation and virulence. Such morphological defects of ssn6/ssn6 mutant were not rescued by overexpression of Tup1, Cph1 or Efg1. Moreover, epistatic analysis showed that, as far as cell morphology was concerned, Ssn6 was epistatic to Tup1 at the higher temperature but that, at the lower temperature, the ssn6/ssn6 tup1/tup1 double mutant grew in a stubby form of pseudohyphae distinct from the phenotypes of either single mutant. Furthermore, overexpression of SSN6 in C. albicans led to enhanced filamentous growth and attenuated virulence. These findings suggest that Ssn6 may function as an activator as well as a repressor of filamentous growth and be a target for candidacidal drugs, as its excess or deficiency resulted in impaired virulence.

• Kozak L et al.
• Elf1p, a member of the ABC class of ATPases, functions as a mRNA export factor in Schizosacchromyces pombe.
• J Biol Chem. 2002; 277: 33580-9
• Display abstract

• Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of the Candida albicans elf1 gene. The elf1 of S. pombe is not an essential gene. When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe.

• Ohi MD, Link AJ, Ren L, Jennings JL, McDonald WH, Gould KL
• Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.
• Mol Cell Biol. 2002; 22: 2011-24
• Display abstract

• Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

• Zhao J, Jin SB, Bjorkroth B, Wieslander L, Daneholt B
• The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm.
• EMBO J. 2002; 21: 1177-87
• Display abstract

• The DEAD box RNA helicase Dbp5 is essential for nucleocytoplasmic transport of mRNA-protein (mRNP) complexes. Dbp5 is present mainly in the cytoplasm and is enriched at the cytoplasmic side of nuclear pore complexes (NPCs), suggesting that it acts in the late part of mRNP export. Here, we visualize the assembly and transport of a specific mRNP particle, the Balbiani ring mRNP in the dipteran Chironomus tentans, and show that a Dbp5 homologue in C.tentans, Ct-Dbp5, binds to pre-mRNP co-transcriptionally and accompanies the mRNP to and through the nuclear pores and into the cytoplasm. We also demonstrate that Ct-Dbp5 accumulates in the nucleus and partly disappears from the NPC when nuclear export of mRNA is inhibited. The fact that Ct-Dbp5 is present along the exiting mRNP fibril extending from the nuclear pore into the cytoplasm supports the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. Finally, the addition of the export factor Dbp5 to the growing transcript highlights the importance of the co-transcriptional loading process in determining the fate of mRNA.

• Zenklusen D, Vinciguerra P, Wyss JC, Stutz F
• Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p.
• Mol Cell Biol. 2002; 22: 8241-53
• Display abstract

• Yra1p/REF participates in mRNA export by recruiting the export receptor Mex67p to messenger ribonucleoprotein (mRNP) complexes. Yra1p also binds Sub2p, a DEAD box ATPase/RNA helicase implicated in splicing and required for mRNA export. We identified genetic and physical interactions between Yra1p, Sub2p, and Hpr1p, a protein involved in transcription elongation whose deletion leads to poly(A)(+) RNA accumulation in the nucleus. By chromatin immunoprecipitation (ChIP) experiments, we show that Hpr1p, Sub2p, and Yra1p become associated with active genes during transcription elongation and that Hpr1p is required for the efficient recruitment of Sub2p and Yra1p. The data indicate that transcription and export are functionally linked and that mRNA export defects may be due in part to inefficient loading of essential mRNA export factors on the growing mRNP. We also identified functional interactions between Yra1p and the exosome components Rrp45p and Rrp6p. We show that yra1, sub2, and Deltahpr1 mutants all present defects in mRNA accumulation and that deletion of RRP6 in yra1 mutants restores normal mRNA levels. The data support the hypothesis that an exosome-dependent surveillance mechanism targets improperly assembled mRNPs for degradation.

• Vasara T, Keranen S, Penttila M, Saloheimo M
• Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components.
• Biochim Biophys Acta. 2002; 1590: 27-40
• Display abstract

• The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.

• Libri D, Graziani N, Saguez C, Boulay J
• Multiple roles for the yeast SUB2/yUAP56 gene in splicing.
• Genes Dev. 2001; 15: 36-41
• Display abstract

• The UAP56 gene has been shown to be required for prespliceosome assembly in mammals. We report here the isolation of the Schizosaccharomyces pombe ortholog of this gene by heterologous complementation of a combined PRP40HA(3)/nam8Delta defect in budding yeast. The Saccharomyces cerevisiae ortholog, YDL084w/SUB2, is also able to suppress this defect. We show that SUB2 is involved in splicing in vivo as well as in vitro. Sub2 defective extracts form a stalled intermediate that contains U2snRNP and can be chased into functional spliceosomes. Our experiments also suggest a role for this protein in events that precede prespliceosome formation. Data reported here as well as in the accompanying papers strongly implicate Sub2p in multiple steps of the spliceosome assembly process.

• Schepers A, Diffley JF
• Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.
• J Mol Biol. 2001; 308: 597-608
• Display abstract

• The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.

• Zenklusen D, Vinciguerra P, Strahm Y, Stutz F
• The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p.
• Mol Cell Biol. 2001; 21: 4219-32
• Display abstract

• Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

• Nakanishi H, Nakayama K, Yokota A, Tachikawa H, Takahashi N, Jigami Y
• Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum.
• Yeast. 2001; 18: 543-54
• Display abstract

• The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.

• Gerber J, Muhlenhoff U, Hofhaus G, Lill R, Lisowsky T
• Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.
• J Biol Chem. 2001; 276: 23486-91
• Display abstract

• Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

• Matern HT, Yeaman C, Nelson WJ, Scheller RH
• The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells.
• Proc Natl Acad Sci U S A. 2001; 98: 9648-53
• Display abstract

• The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.

• Terbush DR, Guo W, Dunkelbarger S, Novick P
• Purification and characterization of yeast exocyst complex.
• Methods Enzymol. 2001; 329: 100-10

• Wilkinson CR, Ferrell K, Penney M, Wallace M, Dubiel W, Gordon C
• Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised.
• J Biol Chem. 2000; 275: 15182-92
• Display abstract

• Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.

• Stutz F et al.
• REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export.
• RNA. 2000; 6: 638-50
• Display abstract

• Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.

• Strasser K, Hurt E
• Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export.
• EMBO J. 2000; 19: 410-20
• Display abstract

• Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

• Takahashi K, Saitoh S, Yanagida M
• Application of the chromatin immunoprecipitation method to identify in vivo protein-DNA associations in fission yeast.
• Sci STKE. 2000; 2000: 1-1
• Display abstract

• The chromatin immunoprecipitation (ChIP) method provides an ideal tool for detecting direct or indirect interactions between proteins of interest and DNAs with known sequences. Here, we introduce the ChIP protocol used in our laboratory to identify in vivo protein-DNA association in the fission yeast Schizosaccharomyces pombe. The cytological and genetic merits of the fission yeast for studying control of the eukaryotic cell cycle and chromosome dynamics are reinforced by application of this ChIP method.

• Guo W, Grant A, Novick P
• Exo84p is an exocyst protein essential for secretion.
• J Biol Chem. 1999; 274: 23558-64
• Display abstract

• The exocyst is a multiprotein complex that plays an important role in secretory vesicle targeting and docking at the plasma membrane. Here we report the identification and characterization of a new component of the exocyst, Exo84p, in the yeast Saccharomyces cerevisiae. Yeast cells depleted of Exo84p cannot survive. These cells are defective in invertase secretion and accumulate vesicles similar to those in the late sec mutants. Exo84p co-immunoprecipitates with the exocyst components, and a portion of the Exo84p co-sediments with the exocyst complex in velocity gradients. The assembly of Exo84p into the exocyst complex requires two other subunits, Sec5p and Sec10p. Exo84p interacts with both Sec5p and Sec10p in a two-hybrid assay. Overexpression of Exo84p selectively suppresses the temperature sensitivity of a sec5 mutant. Exo84p specifically localizes to the bud tip or mother/daughter connection, sites of polarized secretion in the yeast S. cerevisiae. Exo84p is mislocalized in a sec5 mutant. These studies suggest that Exo84p is an essential protein that plays an important role in polarized secretion.

• Schmitt C et al.
• Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.
• EMBO J. 1999; 18: 4332-47
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• Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

• Marston NJ, Richards WJ, Hughes D, Bertwistle D, Marshall CJ, Ashworth A
• Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.
• Mol Cell Biol. 1999; 19: 4633-42
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• Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.

• Lambertson D, Chen L, Madura K
• Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.
• Genetics. 1999; 153: 69-79
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• Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.

• Mamoun CB, Beckerich JM, Gaillardin C, Kepes F
• Disruption of YHC8, a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation.
• J Biol Chem. 1999; 274: 11296-302
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• Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations. Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking. The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum. Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species. The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway. We have identified five homologues of the TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha. We generated a null mutation in the S. cerevisiae YHC8 gene, the closest homologue to Y. lipolytica TSR1, and used different soluble (carboxypeptidase Y, alpha-factor, invertase) and membrane (dipeptidyl-aminopeptidase) secretory proteins to study its phenotype. A large accumulation of soluble protein precursors was detected in the mutant strain. Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum. We propose that the YHC8 gene is a new and important component of the S. cerevisiae endoplasmic reticulum membrane and that it functions in protein translocation/insertion of secretory proteins through or into this compartment.

• Saito A et al.
• cDNA cloning and functional analysis of p44.5 and p55, two regulatory subunits of the 26S proteasome.
• Gene. 1997; 203: 241-50
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• We have employed cDNA cloning to deduce the complete primary structures of p44.5 and p55, two subunits of PA700, a 700-kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consist of 422 and 456 amino acids with calculated molecular masses of 47463 and 52903, and isoelectric points of 6.06 and 7.56, respectively. Computer-assisted homology analysis revealed high sequence similarities of p44.5 and p55 with yeast proteins whose functions are yet unknown. Disruption of the yeast genes, termed NAS4 and NAS5 (non-ATPase subunits 4 and 5), resulted in lethality, indicating that each of the two subunits is essential for proliferation of yeast cells.

• Olkkonen VM, Stenmark H
• Role of Rab GTPases in membrane traffic.
• Int Rev Cytol. 1997; 176: 1-85
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• Small GTPases of the Rab subfamily have been known to be key regulators of intracellular membrane traffic since the late 1980s. Today this protein group amounts to more than 40 members in mammalian cells which localize to distinct membrane compartments and exert functions in different trafficking steps on the biosynthetic and endocytic pathways. Recent studies indicate that cycles of GTP binding and hydrolysis by the Rab proteins are linked to the recruitment of specific effector molecules on cellular membranes, which in turn impact on membrane docking/fusion processes. Different Rabs may, nevertheless, have slightly different principles of action. Studies performed in yeast suggest that connections between the Rabs and the SNARE machinery play a central role in membrane docking/fusion. Further elucidation of this linkage is required in order to fully understand the functional mechanisms of Rab GTPases in membrane traffic.

• Azad AK, Tani T, Shiki N, Tsuneyoshi S, Urushiyama S, Ohshima Y
• Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.
• Mol Biol Cell. 1997; 8: 825-41
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• Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.

• Lo WS, Raitses EI, Dranginis AM
• Development of pseudohyphae by embedded haploid and diploid yeast.
• Curr Genet. 1997; 32: 197-202
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• Diploid strains of S. cerevisiae are known to develop pseudohyphae in response to starvation for nitrogen. We report that both haploid and diploid yeast grow in a filamentous form when embedded in solid media. This is not a response to starvation, since yeast grown on rich media and overlaid with rich agar grow within the agar as pseudohyphae. While we find that the only element of diploidy required for formation of pseudohyphae in response to nitrogen starvation is the a1/alpha 2 repressor, pseudohyphal development by embedded cells does not require a1/alpha 2. Deletion of BUD 5 prevented the formation of pseudohyphae by embedded cells, suggesting that these structures are the result of ordered filament formation rather than agar penetration. Deletion of STE 12 prevented the formation of pseudohyphae by all cell types, showing that the same signal transduction pathway is used by embedded cells as by those responding to nitrogen starvation. Different cell types of yeast thus form filaments in response to several kinds of environmental stimuli.

• Aalto MK, Jantti J, Ostling J, Keranen S, Ronne H
• Mso1p: a yeast protein that functions in secretion and interacts physically and genetically with Sec1p.
• Proc Natl Acad Sci U S A. 1997; 94: 7331-6
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• The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation. We now describe a third suppressor of sec1-1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

• Javerzat JP, Cranston G, Allshire RC
• Fission yeast genes which disrupt mitotic chromosome segregation when overexpressed.
• Nucleic Acids Res. 1996; 24: 4676-83
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• An interference assay has been devised in Schizosaccharomyces pombe to rapidly identify and clone genes involved in chromosome segregation. Random S.pombe cDNAs were overexpressed from an inducible promoter in a strain carrying an additional, non-essential minichromosome. Overexpression of cDNAs derived from four genes, two known (nda3+and ubc4+, encoding beta-tubulin and a ubiquitin conjugating enzyme, respectively) and two unknown, named mlo2+ and mlo3+ (missegregation & lethal when over expressed) caused phenotypes consistent with a failure to segregate chromosomes. Full overexpression of all four cDNAs was lethal. Cells overexpressing nda3+ and ubc4+ cDNAs arrested with condensed unsegregated chromosomes and cells overexpressing mlo2+ displayed an asymmetric distribution of nuclear chromatin. Sublethal levels of overexpression of nda3+, ubc4+ and mlo2+ cDNAs caused elevated rates of minichromosome loss. A third cDNA mlo3+, displayed no increase in the frequency of minichromosome loss at sublethal levels of overexpression but full overexpression caused a complete failure to segregate chromosomes. Our results confirm the assumption that beta-tubulin overexpression is lethal in S.pombe, implicate ubc4+ in the control of metaphase-anaphase transition in fission yeast and finally identify two new genes, mlo2+and mlo3+, likely to play an important role for chromosome transmission fidelity in mitosis.

• Haarer BK, Corbett A, Kweon Y, Petzold AS, Silver P, Brown SS
• SEC3 mutations are synthetically lethal with profilin mutations and cause defects in diploid-specific bud-site selection.
• Genetics. 1996; 144: 495-510
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• Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.

• Blacketer MJ, Madaule P, Myers AM
• The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.
• Mol Cell Biol. 1994; 14: 4671-81
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• Saccharomyces cerevisiae mutant E124 was selected in a visual screen based on elongated cell shape. Genetic analysis showed that E124 contains two separate mutations, pps1-1 and elm4-1, each causing a distinct phenotype inherited as a single-gene trait. In rich medium, pps1-1 by itself causes increased doubling time but does not affect cell shape, whereas elm4-1 results in a moderate cell elongation phenotype but does not affect growth rate. Reconstructed elm4-1 pps1-1 double mutants display a synthetic phenotype in rich medium including extreme cell elongation and delayed cell separation, both characteristics of pseudohyphal differentiation. The elm4-1 mutation was shown to act as a dominant factor that potentiates pseudohyphal differentiation in response to general nitrogen starvation in a genetic background in which pseudohyphal growth normally does not occur. Thus, elm4-1 allows recognition of, or response to, a pseudohyphal differentiation signal that results from nitrogen limitation. PPS1 was isolated and shown to be a previously undescribed gene coding for a protein similar in amino acid sequence to phosphoribosylpyrophosphate synthase, a rate-limiting enzyme in the biosynthesis of nucleotides, histidine, and tryptophan. Thus, the pps1-1 mutation may generate a nitrogen limitation signal, which when coupled with elm4-1 results in pseudohyphal growth even in rich medium.

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