There are two types of protein glycosylation known to occur in budding yeast, Saccharomyces cerevisiae: N-glycosylation on Asn residues and O-glycosylation (O-mannose glycans) on Ser/Thr residues. They play multiple roles in various cellular contexts, and their biosynthetic pathway has been well characterized. In sharp contrast, we know little about the catabolic pathway for these glycans. For the last decade we have gained knowledge on the catabolic pathways for N-glycans and their precursor (dolichol-linked oligosaccharides) in budding yeast 1-5), while virtually nothing was known for the degradation of O-mannose glycans. We recently discovered that, when yeast is grown in media with mannose as a sole carbon source (“mannose-media”), novel free, unconjugated glycans were formed. Upon detailed structural characterization, it was found that they are free, unconjugated glycans structurally identical to O-mannose glycans (“free O-glycans” or FOGs). With these preliminary data in hand, in this proposal we aimed at clarifying the molecular mechanism by which these FOGs are generated. To this end, we utilized various yeast mutants to provide insight into how these FOGs are generated 6). Generation of the novel FOGs in yeastIn terms of the mechanism how these FOGs are generated, we initially speculated that these “free” O-mannose glycans may be generated either by (1) release of O-mannose glycans from a putative de-O-mannosylating enzyme so-called “endo-O-mannosidase (EOMase) (Figure 1), or alternatively, (2) glycans are elongated by biosynthetic enzymes for O-mannose glycans using a free mannose as an acceptor molecule. To validate these hypotheses, we analyzed FOGs using various genetic mutants, and it was found that, in mutants having defect in the first attachment of O-mannose glycans, significant reduction of the amount of FOGs were observed. Moreover, we did not see the elongation of polymannoses on mannose-derivatives when they are included in the culture media. Based on these results, we favor the former possibility, while, as of now, we still cannot The formation of FOGs is negatively regulated by the general transcription repressor, Cyc8 As the occurrence of FOGs was noted only when yeasts were cultured in mannose-media, we hypothesized that they are under the regulation of glucose-repression. Accordingly, we tested various defective mutants for the glucose-repression, and found that a deletion of CYC8, a transcription factor involved in glucose-repression, resulted in vast increase of FOGs when cultured with mannose-media (Figure 2A) 6). Moreover, cyc8 mutants exhibited unusual growth defect under the mannose-media (Figure 2B) 6), implying that Cyc8 may be involved in the controlled expression of a putative EOMase gene, and that deletion of CYC8 may cause constitutive activation of de-O-mannosylation by EOMase upon culturing in mannose-media, leading to cell wall-defect and growth defect under the mannose-media. Indeed, cyc8 mutants also exhibited a sensitivity towards cell wall-disturbing reagents (Figure 2B) 6). Taken all results together, it was hypothesized that the expression of EOMase gene may be strictly controlled, and its overexpression may cause the excessive de-O-mannosylation of cell wall proteins, leading to the cell wall-defect.Figure 1. Release of FOG by a putative EOMasecompletely exclude the latter one 6).By analyzing various mutants with or without luminal expression of α-mannosidase derived from tomato, we concluded that FOGs are accumulated in the post-Golgi compartment6). This is quite distinct from the case with free N-glycans, most of which are found in the cytosol 1,3). 78Clarification of a novel degradation pathway for O-Man glycans in S. cerevisiaeTadashi SuzukiRIKEN Cluster for Pioneering Research (CPR)
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