PMT3 and PMT4, two new members of the protein‐O‐mannosyltransferase gene family of Saccharomyces cerevisiae

Immervoll, Thomas; Gentzsch, Martina; Tanner, Widmar

doi:10.1002/yea.320111403

Cited by 62 publications

(51 citation statements)

References 22 publications

(22 reference statements)

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“…Analy- ses of in vitro mannosyltransfer from Dol-P-Man to specific synthetic acceptor peptides have shown that the simultaneous deletion of PMT1 and PMT2 results in the loss of Ͼ80% of in vitro O-mannosyltransferase activity, when compared with wild-type yeast (1,24). In addition, in vivo O-mannosylation of the same protein substrates is dramatically decreased in pmt1 and pmt2 mutant strains, such as the cell wall proteins Kre9p, chitinase (Cts1p), Bar1p, Ccw4p, and Ccw5p (14,25).…”

Section: Heteromeric Protein Complexes Between Pmt1 and Pmt2 Subfamilmentioning

confidence: 99%

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Girrbach

Strahl

2003

Journal of Biological Chemistry

134

152

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Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi.Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance in many eukaryotes. In yeasts and fungi, the attachment of O-linked mannosyl residues to proteins of the secretory pathway is essential for cell viability (1). In particular it is indispensable for cell wall integrity and normal cellular morphogenesis (2-4). Impairment of O-mannosylation also affects the stability, localization, and/or proper function of individual proteins (5-10). Furthermore, aberrant O-mannosylation can interfere with the retrograde transport of misfolded proteins across the membrane of the endoplasmic reticulum (ER) 1 (11). O-mannosylation is not only important in yeast, but also in mammals. It was recently shown that in humans, O-mannosyl glycosylation represents a new pathomechanism for muscular dystrophy and neuronal migration disorders (12, 13).In yeast and fungi, O-mannosylation is initiated in the lumen of the ER by an essential family of protein O-mannosyltransferases (PMTs). These enzymes catalyze the transfer of mannose from dolichyl phosphate-activated mannose (Dol-PMan) to serine or threonine residues of secretory proteins (2). In Saccharomyces cerevisiae, a total of seven PMT family members (Pmt1-7p) have been identified, which share almost identical hydropathy profiles that predict the PMTs to b...

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Section: Heteromeric Protein Complexes Between Pmt1 and Pmt2 Subfamilmentioning

confidence: 99%

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Girrbach

Strahl

2003

Journal of Biological Chemistry

134

152

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“…Most interestingly, all of the Pmts share a nearly identical hydropathy profile, wherein an integral membrane protein with a tripartite structure (amino-and carboxyl-terminal regions, each with several putative transmembrane helices, and a central hydrophilic segment) is predicted (6,10,11,15,16). Strikingly, this pattern diverges from structural models of other ER glycosyltransferases as well as from the common type II model of glycosyltransferases of the Golgi apparatus.…”

mentioning

confidence: 94%

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

Strahl‐Bolsinger

Scheinost

1999

Journal of Biological Chemistry

100

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The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino-and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.Glycosylation is one of the most elaborate covalent protein modifications known. The carbohydrate chains can be coupled to the protein through either an N-or O-glycosidic bond. Protein O-mannosylation, originally observed in fungi (1), is initiated at the endoplasmic reticulum by protein mannosyltransferases (Pmts) 1 that catalyze the transfer of a mannosyl residue from dolichyl phosphate-activated mannose (Dol-PMan) to serine or threonine residues of nascent proteins entering the secretory pathway; in the Golgi apparatus additional sugars are added to the O-linked mannose with GDP-mannose serving as carbohydrate donor (2, 3). Dol-P-Man-dependent O-glycosylation of secreted proteins is a general feature of yeasts and filamentous fungi (4).The key enzyme of protein O-mannosylation, the Dol-P-Man: protein O-mannosyltransferase Pmt1p, was purified from Saccharomyces cerevisiae following the enzyme activity, and the corresponding gene was cloned (5, 6). Pmt1p is an integral membrane glycoprotein located at the ER (5, 7-9). Based on homology to Pmt1p, a family of seven protein O-mannosyltransferases (Pmt1p-Pmt7p) has been identified (10 -13). Thus far, protein O-mannosyltransferase activity has been demonstrated for Pmt1p, Pmt2p, Pmt3p, Pmt4p, and Pmt6p (13, 14). The individual mannosyltransferases recognize specific protein substrates that might explain the presence of more than one transferase in S. cerevisiae (14). Moreover, Pmtp orthologues have been identified from other yeasts (4), from the opportunistic fungal pathogen Candida alb...

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“…The N-and C-terminal thirds are lipophilic with multiple potential transmembrane helices, whereas the central part is hydrophilic. Recently, four more PMT genes (PMT3 6) have been detected ( [6,7], Dommaschk et al, unpubl.). *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Protein O‐glycosylation in Saccharomyces cerevisiae: the protein O‐mannosyltransferases Pmt1p and Pmt2p function as heterodimer

1995

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PMT3 and PMT4, two new members of the protein‐O‐mannosyltransferase gene family of Saccharomyces cerevisiae

Cited by 62 publications

References 22 publications

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

Protein O‐glycosylation in Saccharomyces cerevisiae: the protein O‐mannosyltransferases Pmt1p and Pmt2p function as heterodimer

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