Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase.
The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.
Cipollo
John F
JF
Trimble
Robert B
RB
D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D019897:Periplasm / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; C480019:Alg12 protein, S cerevisiae; D008364:Mannosyltransferases; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase; D010450:Endopeptidases