Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species

The genes encoding GDP-mannose pyrophosphorylase from Saccharomyces cerevis iae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the cont rol of the tightly regulated promoters of MET3 and CaMET3 respectively. Nor thern analysis showed that the addition of methionine effectively blocks th e transcription of pMET3-SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methioni ne-mediated repression of CaSRB1 caused loss of viability in C. albicans, d emonstrating that, as in S. cerevisiae, the gene is essential for growth. D epletion of GDP-mannose pyrophosphorylase had a highly pleiotropic effect i n the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selectio n, clumping and flocculation, as well as increased sensitivity to a wide ra nge of antifungal drugs and cell wall inhibitors, and impaired hyphal switc hing ability. These phenotypes resulted from defects in glycosylation, as d emonstrated by reduced affinity for Alcian blue and sensitivity to hygromyc in B. Our results provide new information about the roles of protein glycos ylation in yeast and, in particular, the steps that require GDP-mannose in the fungal pathogen C. albicans.