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 cerevisiae sec4-8 mutation, and its deduced protein product (Sec4p) was 63% identical 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 an amino acid substitution (Ser-28-->Asn) analogous to the Ser-17-->Asn substitution in a trans-dominant inhibitor of mammalian Ras protein. GAL1-regulated expression plasmids carrying the mutant sec4 allele (pS28N) had minimal effects in glucose-incubated C. albicans transformants, but six of nine transformants tested grew very slowly in galactose. Incubation of pS28N transformants in galactose also inhibited secretion of aspartyl protease (Sap) and caused 90-nm secretory vesicles to accumulate intracellularly, and plasmid curing restored growth and Sap secretion to wild-type levels. These results imply that C. albicans SEC4 is required for growth and protein secretion and that it functions at a later step in the protein secretion pathway than formation of post-Golgi secretory vesicles. They also demonstrate the feasibility of using inducible dominant-negative alleles to define the functions of essential genes in C. albicans.
Mao
Y
Y
Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.
Kalb
V F
VF
Wong
B
B
D000483:Alleles; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000166:cytology / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D002455:Cell Division / Q000235:genetics; D005690:Galactose / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D005799:Genes, Dominant; D005947:Glucose / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*
D029701:Saccharomyces cerevisiae Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D005947:Glucose; D005690:Galactose