Abstract
In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans (i.e. free N-glycans) are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum-associated degradation or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of free N-glycans is now well-understood, but the issue of whether other types of free glycans are present remains unclear. Here, we report on the accumulation of free, O-mannosylated glycans in budding yeast that were cultured in medium containing mannose as the carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, which encodes for a general transcription repressor, resulted in the accumulation of excessive amounts of free O-glycans, concomitant with a severe growth defect, a reduction in the level of an O-mannosylated protein, and compromised cell wall integrity. Our findings provide evidence in support of a regulated pathway for the degradation of O-glycoproteins in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.
Highlights
In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum–associated degradation or from lipidlinked oligosaccharides, donor substrates for N-glycosylation of proteins
The generation and catabolism of free oligosaccharides derived from glycoproteins as well as linked oligosaccharides (LLOs) are fundamental phenomena that occur from bacteria to mammalian cells [17, 18, 32]
We serendipitously discovered that a series of novel free glycans that are structurally related to O-mannosyl glycans are produced only when the yeast cells are cultured in media containing mannose as the sole carbon source
Summary
The findings reported in our previous studies suggested that the yeast cytosolic/vacuolar ␣-mannosidase, Ams, plays a pivotal role in the catabolism of FNGs that accumulate in the cytosol [22, 25, 27]. The generation of FNGs was reduced when certain types of culture media, such as YPGal, YPGly, and YP, were used, probably due to the intracellular activation of Ams. The generation of FNGs was reduced when certain types of culture media, such as YPGal, YPGly, and YP, were used, probably due to the intracellular activation of Ams1 This result is consistent with previous observations that Ams activity is increased when the level of glucose in the culture media is reduced [25, 27]. The oligomers corresponding to these peaks were structurally altered on treatment with jack bean ␣-mannosidase (Fig. 1B), suggesting that they are ␣-mannosylated glycans These results suggest that, in budding yeast, the generation of novel ␣-mannosylated free oligosaccharides can be induced by using YPMan as a medium
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