Letter to the Glyco-Forum. Hypoglycosylation in the alg12Δ yeast mutant destabilizes protease A and causes proteolytic loss of external invertase

Abstract

The Saccharomyces cerevisiae alg12Δ mutant accumulates oligosaccharide lipid with a Man7GlcNAc2 oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12Δ strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12Δ background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9Δ invertase was more severely hypoglycosylated than the alg12Δ 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 alg12Δ yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9Δ invertase is under-glycosylated in comparison to the alg12Δ form, it is more stable because in this genetic background much less protease A is secreted compared to alg12Δ cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, α-glucosidase) as probes when characterizing glycosylation defects in yeast.