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

Abstract

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...

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

“…Yeast strains were grown under standard conditions and transformed following the method of Gietz et al (27) with the yeast shuttle vectors pRS423 (28), YEp352 (29), pSB53 (19), pSB56 (18), PMT2-YEp352 (23), pVG13 (18), pSB114 (18), and the plasmids listed below. Standard procedures were used for all DNA manipulations (30).…”

“…Its N terminus faces the cytoplasm whereas the C terminus faces the lumen of the ER. Two major hydrophilic domains that are located between transmembrane spans one and two (loop 1) and transmembrane spans five and six (loop 5), respectively, are oriented toward the ER lumen and are essential for Pmt1p activity (18,19). The replacement of invariant amino acid residues in these regions suggested that these segments are involved in the recognition and/or binding of protein substrates and/or catalysis (18).…”

“…Pmt1p is an integral ER membrane glycoprotein with seven transmembrane-spanning domains (19). Its N terminus faces the cytoplasm whereas the C terminus faces the lumen of the ER.…”

TheO-Mannosyltransferase PMT4 Is Essential for Normal Appressorium Formation and Penetration inUstilago maydis

“…Hydropathy analysis (Kyte and Doolittle, 1982) of Um11220, Um10749, and Um05433 suggest that they conserve the characteristic structure of O-mannosyltransferase proteins of two large transmembrane domains, located in their N-and C-terminal regions and separated by a central hydrophilic region (Strahl-Bolsinger and Scheinost, 1999). In addition to the topological distribution, the putative U. maydis PMT proteins conserve the number of predicted transmembrane domains with respect to S. cerevisiae PMTs: 11 for Um11220 and nine for Um10749 and Um05433 (see Supplemental Figure 1 online).…”

Membrane association is a determinant for substrate recognition by PMT4 protein O -mannosyltransferases

“…PMTs have been identified and extensively characterized in yeast. In Saccharomyces cerevisiae, seven PMT family members (Pmt1p to -7p) are present (8,9) that are integral ER membrane proteins with seven transmembranespanning domains (12). Phylogenetic analyses indicate that the PMT family is subdivided into the PMT1, PMT2, and PMT4 subfamilies, whose members include transferases closely related to S. cerevisiae Pmt1p, Pmt2p, and Pmt4p, respectively (1,13).…”