Diminished activity of the first N-glycosylation enzyme, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), gives rise to mutant phenotypes in yeast

Abstract

The enzyme which initiates the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT), is encoded by the ALG7 gene. Essential for viability, ALG7 has been evolutionarily conserved and shown to be involved in a variety of functions. ALG7 is an early growth-response gene in yeast, and downregulation of ALG7 expression results in diminished N-glycosylation and secretion of Xenopus oocyte proteins. We have now investigated the consequences of diminished GPT activity in yeast using mutant ALG7 genes with deletions in the 3′ untranslated region (3′ UTR). We show that a 2.5- to 4-fold reduction in GPT activity gave rise to distinct phenotypes, whose severity was inversely related to the level of GPT activity. These phenotypes included hypersensitivity to tunicamycin, enlarged cell size, extensive aggregation, lack of a typical stationary (G 0) arrest, and defective spore germination. We conclude that yeast cells are sensitive to GPT dosage, and that attenuation of GPT activity interferes with various functions in the yeast life cycle.