Abstract
In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.
Highlights
Yeast free oligosaccharides are cleaved from glycoproteins during endoplasmic reticulum (ER)-associated degradation of proteins (ERAD), but factors underlying their control remain to be explored
A wild type yeast population doubles about every 90 min until the glucose required for fermentation is consumed (30)
In order to look more closely at these questions, we characterized free oligosaccharides (fOS) generated in ams1⌬ cells and examined how the different structures are modulated in cells deficient in various gene products involved in ER-associated degradation of proteins (ERAD)-L. Cells, indicating that this pathway is not involved in fOS generation under our growth conditions (Fig. 4C). These results indicate that only about one-half of fOS can be attributed to known ERAD processes and that, of the ERAD pathways, ERAD-L is the major source of fOS
Summary
Yeast free oligosaccharides (fOS) are cleaved from glycoproteins during ER-associated degradation of proteins (ERAD), but factors underlying their control remain to be explored. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man7GlcNAc2 (d1) N-glycans with non-reducing ␣1,6-mannosyl residues Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). We demonstrate that both Png1p-dependent generation of Man7–10GlcNAc2 fOS and vacuolar ␣-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man1GlcNAc2 are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. A fOS possessing the Och1p-dependent d4 mannose residue as its sole terminal non-reducing ␣1,6-mannosyl motif was found to be highly regulated by Yos9p, our data cannot distinguish between lectin-dependent and lectin-independent roles for Yos9p during ERAD
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