Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae.

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammalsO-mannosylation is the only type ofO-glycosylation. In an essential step toward the full understanding of proteinO-mannosylation we mapped theO-mannose glycoproteome in baker's yeast. Taking advantage of anO-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500O-glycoproteins from all subcellular compartments for which over 2300O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized proteinO-mannosyltransferases. We find thatO-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed thatO-mannosylation is favored in unstructured regions and ß-strands. Furthermore,O-mannosylation is impeded in the proximity ofN-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and theirO-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types ofO-glycosylation from yeast to mammals.

Protein O-mannosyltransferases associate with the translocon to modify translocating polypeptide chains.

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.

Photoaffinity labeling of protein O-mannosyltransferases of the PMT1/PMT2 subfamily.

Protein O-mannosylation is initiated at the endoplasmic reticulum (ER) by dolichyl phosphate-mannose: protein O-mannosyltransferases (PMTs). PMTs are members of the glycosyltransferase (GT) C superfamily. They are large polytopic integral membrane proteins located in the ER membrane. PMTs utilize dolichyl phosphate--activated mannose as sugar donor. Glycosyltransfer of mannose to serine and threonine residues of nascent polypeptides leads to an inversion of the stereochemistry of the glycosidic bond. Here, we describe photoaffinity labeling of yeast Pmt1p using a photo-reactive probe that is based on the artificial mannosyl acceptor peptide YATAV. Due to the high homology of PMTs, this method can also be applied to study PMT1 and PMT2 subfamily members from fungi other than baker's yeast.

Protein O-mannosylation: what we have learned from baker's yeast.

BACKGROUND: Protein O-mannosylation is a vital type of glycosylation that is conserved among fungi, animals, and humans. It is initiated in the endoplasmic reticulum (ER) where the synthesis of the mannosyl donor substrate and the mannosyltransfer to proteins take place. O-mannosylation defects interfere with cell wall integrity and ER homeostasis in yeast, and define a pathomechanism of severe neuromuscular diseases in humans. SCOPE OF REVIEW: On the molecular level, the O-mannosylation pathway and the function of O-mannosyl glycans have been characterized best in the eukaryotic model yeast Saccharomyces cerevisiae. In this review we summarize general features of protein O-mannosylation, including biosynthesis of the mannosyl donor, characteristics of acceptor substrates, and the protein O-mannosyltransferase machinery in the yeast ER. Further, we discuss the role of O-mannosyl glycans and address the question why protein O-mannosylation is essential for viability of yeast cells. GENERAL SIGNIFICANCE: Understanding of the molecular mechanisms of protein O-mannosylation in yeast could lead to the development of novel antifungal drugs. In addition, transfer of the knowledge from yeast to mammals could help to develop diagnostic and therapeutic approaches in the frame of neuromuscular diseases. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

C terminus of Nce102 determines the structure and function of microdomains in the Saccharomyces cerevisiae plasma membrane.

The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.

Plasma membrane microdomains regulate turnover of transport proteins in yeast.

In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.