Overexpression of a dominant-negative allele of YPT1 inhibits growth and aspartyl protease secretion in Candida albicans

To investigate the pre-Golgi secretion pathway in the pathogenic yeast Cand ida albicans we cloned the C. albicans homologue of the Saccharomyces cerev isiae protein secretion gene YPT1. The C. albicans YPT1 ORF contained a 624 bp intronless ORF encoding a deduced protein of 207 aa and 2.3 kDa. This d educed protein was 77% identical to S. cerevisiae Ypt1 protein (Ypt1p) and it contained GTP-binding domains that are conserved among all known ras-lik e GTPases. Multicopy plasmids containing C. albicans YPT1 complemented the temperature-sensitive S. cerevisiae ypt1 (A136D) mutation. One chromosomal YPT1 allele in C. albicans CA14 was readily disrupted by homologous gene ta rgeting, but attempts to disrupt the second allele yielded no viable null m utants. Since this suggested that C. albicans YPT1 may be essential, a muta nt ypt1 allele was constructed encoding the amino acid substitution analogo us to the N1211 substitution in a known trans-dominant inhibitor of S. cere visiae Ypt1p. Next, a GAL1-regulated plasmid was used to express the mutant ypt1(N121I) allele in C. albicans CAI4. Ten of 11 transformants tested gre w normally in glucose and poorly in galactose, and plasmid curing restored growth to wild-type levels. When these transformants were incubated in gala ctose, secretion of aspartyl proteinase (Sap) was inhibited and membrane-bo und secretory vesicles accumulated intracellularly. These results imply tha t C. albicans YPT1 is required for growth and protein secretion, and they c onfirm the feasibility of using inducible dominant-negative alleles to defi ne the functions of essential genes in C. albicans.