Sabine Strahl-Bolsinger

Protein O-mannosylation

Protein O-mannosylation, originally observed in fungi, starts at the endoplasmic reticulum with the transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins. This reaction is catalyzed by a family of protein O-mannosyltransferases (PMTs), which were first characterized in Saccharomyces cerevisiae. The identification of this evolutionarily conserved PMT gene family has led to the finding that protein O-mannosylation plays an essential role in a number of physiologically important processes. Focusing on the PMT gene family, we discuss here the main aspects of the biogenesis of O-linked carbohydrate chains in S. cerevisiae, Candida albicans, and other fungi. We summarize recent work utilizing pmt mutants that demonstrates the impact of protein O-mannosylation on protein secretion, on maintenance of cell wall integrity, and on budding. Further, the occurrence of PMT orthologs in higher eukaryotes such as Arabidopsis, Drosophila and mammals is reported and discussed.

Morphogenesis, Adhesive Properties, and Antifungal Resistance Depend on the Pmt6 Protein Mannosyltransferase in the Fungal Pathogen Candida albicans

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.

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

The identification of the evolutionarily conserved family of dolichyl-phosphate-d-mannose:proteinO- 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, fromSaccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenousN-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.