In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co-or posttranslational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-␤-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole ␣-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

Background:The enzyme generating free oligosaccharides (fOSs) in the lumen of the endoplasmic reticulum (ER) has been unidentified. Results: Oligosaccharyltransferase (OST), the N-glycosylating enzyme, hydrolyzes dolichol-linked oligosaccharides to release the fOSs. Conclusion: OST is responsible for the generation of fOSs in the ER lumen. Significance: This study provides a mechanistic insight into the formation of luminal fOSs in yeast.

In Saccharomyces cerevisiae, protein O-mannosylation, which is executed by protein O-mannosyltransferases, is essential for a variety of biological processes as well as for conferring solubility to misfolded proteins. To determine if O-mannosylation plays an essential role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, we used a model misfolded protein, Gas1*p. The O-mannose content of Gas1*p, which is transferred by protein O-mannosyltransferases, was higher than that of Gas1p. Both Pmt1p and Pmt2p, which do not transfer O-mannose to correctly folded Gas1p, participated in the O-mannosylation of Gas1*p. Furthermore, in a pmt1 Delta pmt2 Delta double-mutant background, degradation of Gas1*p is altered from a primarily proteasome dependent to a vacuolar protease-dependent pathway. This process is in a manner dependent on a Golgi-to-endosome sorting function of the VPS30 complex II. Collectively, our data suggest that O-mannosylation plays an important role for proteasome-dependent degradation of Gas1*p via the ERAD pathway and when O-mannosylation is insufficient, Gas1*p is degraded in the vacuole. Thus, we propose that O-mannosylation by Pmt1p and Pmt2p might be a key step in the targeting of some misfolded proteins for degradation via the proteasome-dependent ERAD pathway.

Asparagine (N)-linked protein glycosylation, which takes place in the eukaryotic endoplasmic reticulum (ER), is important for protein folding, quality control and the intracellular trafficking of secretory and membrane proteins. It is known that, during N-glycosylation, considerable amounts of lipid-linked oligosaccharides (LLOs), the glycan donor substrates for N-glycosylation, are hydrolyzed to form free N-glycans (FNGs) by unidentified mechanisms. FNGs are also generated in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins during ER-associated degradation. FNGs derived from LLOs and misfolded glycoproteins are eventually merged into one pool in the cytosol and the various glycan structures are processed to a near homogenous glycoform. This article summarizes the current state of our knowledge concerning the formation and catabolism of FNGs.