Transmembrane topology of Pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases

The identification of the evolutionarily conserved family of dolichyl-phosp hate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically impo rtant processes, Strikingly, all members of the Pmt protein family share al most identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative tran smembrane helices, This pattern is of particular interest because it diverg es 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. Struc tural predictions were directly tested by site-directed mutagenesis of endo genous N-glycosylation sites, by fusing a topology-sensitive monitor protei n domain to carboxyl-terminal truncated versions of the Pmt1 protein and, i n 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 b y five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function o f Pmt1p.