Aberrant Processing of the WSC Family and Mid2p Cell Surface Sensors Results in Cell Death of Saccharomyces cerevisiae O-Mannosylation Mutants

Abstract

Protein O mannosylation is a crucial protein modification in uni-and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4⌬ mutant. We found that pmt2 pmt4⌬ cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4⌬ mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4⌬ mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.Protein O mannosylation is initiated at the endoplasmic reticulum (ER) by the transfer of mannose from dolichyl phosphate-activated mannose to serine or threonine residues of secretory proteins (52). This reaction is catalyzed by an essential family of protein O-mannosyltransferases (PMTs) that is evolutionarily conserved from yeast to humans (28,52,57). The PMT family is divided into the PMT1, PMT2, and PMT4 subfamilies, whose members include transferases closely related to Saccharomyces cerevisiae Pmt1p, Pmt2p, and Pmt4p, respectively (17,57). In S. cerevisiae the entire PMT family is highly redundant (Pmt1 to Pmt7p), and members of the PMT1 and PMT2 subfamilies show marked similarities and distinctions from PMT4 subfamily members. For example, members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p), whereas the unique representative of the PMT4 subfamily forms homomeric complexes (16). Further, the PMT1/PMT2 and PMT4 subfamilies use different acceptor protein substrates in vivo (10,14).Studies of pmt mutants revealed that protein O mannosylation plays a substantial role in uni-and multicellular eukaryotes. In humans, mutations in POMT1 (protein O-mannosyltransferase 1 gene), which encodes a putative counterpart of the yeast Pmt4p O-mannosyltransferase, result in Walker-Warburg syndrome, which is characterized by severe congenital muscular dystrophy, a neuronal migration defect, and structural abnormalities of the eye (2). Mutations of the Drosophila POMT1 orth...

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

“…The latter is noteworthy, since the PKC1-dependent cell wall integrity pathway is defective in the O-glycosylation mutants impaired in the activity of protein-O-mannosyl transferases (PMTs) (Strahl-Bolsinger et al, 1999). In these mutants, Mpk1p (Slt2) phosphorylation could be restored in part by overexpression of proteins acting upstream of the pathway, such as the cell wall sensors from the Wsc family and Mid2p (Lommel et al, 2004). Under our experimental conditions, however, despite the general defect of glycosylation ascribed to sec59-1 or dpm1-6 mutants, the Mpk1/Slt2p phosphorylation is still observed.…”

The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans

“…A possible explanation could be the alteration of the sensors that activate the Mkc1p pathway. Recently, in S. cerevisiae, it has been shown that the incorrect Omannosylation of the Wsc1p, Wsc2p and Mid2p receptors in pmt2D pmt4D mutants prevents the activation of Slt2p upon exposure to elevated temperature (Lommel et al, 2004). In C. albicans, Hog1p might control the expression of the proteins involved in glycosylation of the receptors that activate the Mkc1p MAP kinase pathway.…”

Comparative proteomic analysis of an Aspergillus fumigatus mutant deficient in glucosidase I (AfCwh41)

“…Wsc1 and Mid2 serve a partially overlapping role in detecting and transmitting cell wall status to Rho1. N-or O-glycans of these two molecules are important for their function (Lommel et al, 2004;Philip & Levin, 2001;Hutzler et al, 2008). Upon environmental stimuli or cell wall stress, Rho1 is activated, which then activates downstream effectors such as the Pkc1-MAP kinase cascade, b-1,3-glucan synthase and Bni1/ SepA formin protein (Walther & Wendland, 2003;Longtine & Bi, 2003;Guest et al, 2004).…”