Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans

Candida albicans SEC4 was cloned by complementing the Saccharomyces cerevis iae sec4-8 mutation, and its deduced protein product (Sec4p) was 63% identi cal to S. cerevisiae Sec4p. One chromosomal SEC4 allele in C, albicans CAI4 was readily disrupted by homologous gene targeting, but efforts to disrupt the second allele yielded no viable null mutants. Although this suggested that C. albicans SEC4 was essential, it provided no information about this gene's functions. Therefore, we constructed a mutant sec4 allele encoding a n amino acid substitution (Ser-28-->Asn) analogous to the Ser-17-->Asn subs titution in a trans-dominant inhibitor of mammalian Ras protein, GAL1-regul ated expression plasmids carrying the mutant sec4 allele (pS28N) had minima l effects in glucose-incubated C. albicans transformants, but six of nine t ransformants tested grew very slowly in galactose. Incubation of pS28N tran sformants in galactose also inhibited secretion of aspartyl protease (Sap) and caused 90-nm secretory vesicles to accumulate intracellularly, and plas mid curing restored growth and Sap secretion to wild-type levels, These res ults imply that C, albicans SEC4 is required for growth and protein secreti on and that it functions at a later step in the protein secretion pathway t han formation of post-Golgi secretory vesicles. They also demonstrate the f easibility of using inducible dominant-negative alleles to define the funct ions of essential genes in C. albicans.