1887

Abstract

To investigate the pre-Golgi secretion pathway in the pathogenic yeast , we cloned the homologue of the protein secretion gene . The ORF contained a 624 bp intronless ORF encoding a deduced protein of 207 aa and 23 kDa. This deduced protein was 77% identical to Ypt1 protein (Ypt1p) and it contained GTP-binding domains that are conserved among all known ras-like GTPases. Multicopy plasmids containing complemented the temperature-sensitive (A136D) mutation. One chromosomal allele in CAI4 was readily disrupted by homologous gene targeting, but attempts to disrupt the second allele yielded no viable null mutants. Since this suggested that may be essential, a mutant allele was constructed encoding the amino acid substitution analogous to the N121I substitution in a known -dominant inhibitor of Ypt1p. Next, a -regulated plasmid was used to express the mutant (N121I) allele in CAI4. Ten of 11 transformants tested grew normally in glucose and poorly in galactose, and plasmid curing restored growth to wild-type levels. When these transformants were incubated in galactose, secretion of aspartyl proteinase (Sap) was inhibited and membrane-bound secretory vesicles accumulated intracellularly. These results imply that is required for growth and protein secretion, and they confirm the feasibility of using inducible dominant-negative alleles to define the functions of essential genes in .

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2001-07-01
2019-10-22
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