Secondary literature sources for MIR

The following references were automatically generated.

Maeda Y et al.
PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.
EMBO J. 2001; 20: 250-61
Display abstract
Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis. GPI-MT-I, however, has not been molecularly characterized. Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M. PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S
Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p.
J Biol Chem. 2000; 275: 19288-96
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Protein O-mannosylation is an essential protein modification. It is initiated at the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to humans. Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the endoplasmic reticulum. ScPmt1p forms a complex with ScPmt2p that is required for maximum transferase activity. Recently, we proposed a seven-transmembrane structural model for ScPmt1p. A large, hydrophilic, endoplasmic reticulum-oriented segment is flanked by five amino-terminal and two carboxyl-terminal membrane-spanning domains. Based on this model, a structure-function analysis of ScPmt1p was performed. Deletion mutagenesis identified the N-terminal third of the transferase as being essential for the formation of a functional ScPmt1p-ScPmt2p complex. Deletion of the central hydrophilic loop eliminates mannosyltransferase activity, but not ScPmt1p-ScPmt2p interactions. Alignment of all fully characterized PMT family members revealed that this central loop region contains three highly conserved peptide motifs, which can be considered as signatures of the PMT family. In addition, a number of invariant amino acid residues were identified throughout the entire protein sequence. In order to evaluate the functional significance of these conserved residues site-directed mutagenesis was performed. We show that several amino acid substitutions in the conserved motifs significantly reduce ScPmt1p activity. Further, the invariant residues Arg-64, Glu-78, Arg-138, and Leu-408 are essential for ScPmt1p function. In particular, Arg-138 is crucial for ScPmt1p-ScPmt2p complex formation.

Todorow Z, Spang A, Carmack E, Yates J, Schekman R
Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.
Proc Natl Acad Sci U S A. 2000; 97: 13643-8
Display abstract
Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.

Distel B, Braakman I, Elgersma Y, Tabak HF
Transactions at the peroxisomal membrane.
Subcell Biochem. 2000; 34: 303-22

Kojima H, Hashimoto H, Yoda K
Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae.
Biosci Biotechnol Biochem. 1999; 63: 1970-6
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Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.

Burda P, Jakob CA, Beinhauer J, Hegemann JH, Aebi M
Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.
Glycobiology. 1999; 9: 617-25
Display abstract
The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.

Jungmann J, Rayner JC, Munro S
The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex.
J Biol Chem. 1999; 274: 6579-85
Display abstract
In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.

Gentzsch M, Tanner W
Protein-O-glycosylation in yeast: protein-specific mannosyltransferases.
Glycobiology. 1997; 7: 481-6
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S. cerevisiae contains at least six genes (PMT1-6) for dolicholphosphate-D-mannose: protein-O-D-mannosyltransferases. The in vivo mannosylation of seven O-mannosylated yeast proteins has been analyzed in a number of pmt mutants. The results clearly indicate that the various protein O-mannosyltransferases have different specificities for protein substrates. Five of the proteins tested (chitinase, a-agglutinin, Kre9p, Bar1p, Pir2p/hsp 150) are mainly underglycosylated in pmt1 and pmt2 mutants, whereby qualitative differences exist among the various proteins. Two of the O-mannosylated proteins (Ggp1p and Kex2p) are not at all affected in pmt1 and pmt2 mutants but are clearly underglycosylated when PMT4 is mutated. Although the PMT4 gene product is shown to be responsible for O-mannosylating a Ser-rich region of Ggp1p in vivo, a penta-seryl-peptide is not an in vitro substrate for this transferase. A PMT3 mutation does affect O-mannosylation of chitinase only in the genetic background of a pmt1pmt2 double mutation, indicating that PMT1 and PMT2 can compensate for a deleted PMT3 gene.

Gentzsch M, Tanner W
The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital.
EMBO J. 1996; 15: 5752-9
Display abstract
The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.

Immervoll T, Gentzsch M, Tanner W
PMT3 and PMT4, two new members of the protein-O-mannosyltransferase gene family of Saccharomyces cerevisiae.
Yeast. 1995; 11: 1345-51
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Two genes PMT3 and PMT4 were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genes PMT1, PMT2 of Saccharomyces cerevisiae and part of a PMT1 related sequence of Kluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate-D-mannose (Dol-P-Man) to seryl and threonyl residues in proteins. The products encoded by the PMT3 and PMT4 genes have almost identical hydropathy profiles in comparison to PMT1 and PMT2: a hydrophobic N- and C-terminal third each with multiple potential transmembrane helices and a central hydrophilic part. The predicted Pmt3p contains 753 amino acids, four potential N-glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N-glycosylation sites. Northern blot analysis showed a single mRNA transcript of PMT3 and PMT4 of 2.8 kb. Thus PMT3 and PMT4 are two new members of the PMT gene family. The pmt4 null mutant the pmt3 pmt4 double null mutant, but not pmt3 null mutant, showed a significant shift of chitinase due to under glycosylation of the enzyme. The triple disruption pmt2 pmt3 pmt4 and the quadruple disruption result in a lethal phenotype.

Gentzsch M, Immervoll T, Tanner W
Protein O-glycosylation in Saccharomyces cerevisiae: the protein O-mannosyltransferases Pmt1p and Pmt2p function as heterodimer.
FEBS Lett. 1995; 377: 128-30
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The protein O-mannosyltransferases Pmt1p and Pmt2p are catalyzing the O-glycosylation of serine and threonine residues in the endoplasmic reticulum of yeast. Deletion of each of these proteins by disruption of the corresponding gene leads to a dramatic decrease of mannosyltransferase activity in vitro. With an anti-Pmt1p immunoaffinity column a complex of Pmt1p and a second protein was purified; this protein turned out to be Pmt2p. Overexpression of Pmt1p or Pmt2p, respectively, does not increase mannosyltransferase activity in vitro. Overexpression of both mannosyltransferases together, however, raises in vitro activity threefold. These data indicate that Pmt1p and Pmt2p function as a complex catalyzing protein O-glycosylation in yeast.

Lussier M, Gentzsch M, Sdicu AM, Bussey H, Tanner W
Protein O-glycosylation in yeast. The PMT2 gene specifies a second protein O-mannosyltransferase that functions in addition to the PMT1-encoded activity.
J Biol Chem. 1995; 270: 2770-5
Display abstract
The PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D.B., and Bussey, H. (1993) Genome 36, 32-42). The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S. cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8164-8168). The PMT2 gene encodes a new protein O-D-mannosyltransferase. Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.

Zimmerman JW, Robbins PW
The hydrophobic domain of dolichyl-phosphate-mannose synthase is not essential for enzyme activity or growth in Saccharomyces cerevisiae.
J Biol Chem. 1993; 268: 16746-53
Display abstract
Dolichyl-phosphate-mannose synthase is a membrane-bound enzyme of the endoplasmic reticulum that catalyzes the formation of dolichyl phosphate mannose from dolichyl phosphate and GDP-mannose. It is an essential enzyme for growth of Saccharomyces cerevisiae, and, like other enzymes that utilize some form of the lipid dolichol as substrate, dolichyl-phosphate-mannose synthase contains a putative "dolichol recognition sequence" in the predicted membrane-spanning domain. To investigate the importance of this sequence in particular and the hydrophobic region in general, a series of mutants of dolichyl-phosphate-mannose synthase were constructed that contained successive deletions or mutations of the hydrophobic region, and their in vivo functions and in vitro activities were examined. While all of the mutant proteins exhibited decreased transferase activities in vitro compared to the wild-type enzyme, the sequence was not essential for growth or for protein glycosylation in S. cerevisiae. Interestingly, although deletion of the entire hydrophobic region resulted in a soluble protein, mutant proteins containing 3 or 8 hydrophobic residues at the carboxyl terminus were still membrane-associated. These mutant proteins could be released from membranes by treatment with sodium carbonate, indicating peripheral associations.

Strahl-Bolsinger S, Immervoll T, Deutzmann R, Tanner W
PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae.
Proc Natl Acad Sci U S A. 1993; 90: 8164-8
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The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity. The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway. This type of mannosyl transfer reaction has so far been observed only in fungal cells. Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library. A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb. The predicted protein consists of 817 amino acids including three potential N-glycosylation sites. The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part. Expression of the clone in Escherichia coli resulted in mannosyltransferase activity. Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay. In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay.

Strahl-Bolsinger S, Tanner W
Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase.
Eur J Biochem. 1991; 196: 185-90
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The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.