Overexpression of a dominant-negative allele of YPT1 inhibits growth and aspartyl protease secretion in Candida albicans The GenBank accession number for the C. albicans YPT1 sequence reported in this paper is AF330211.

Abstract

To investigate the pre-Golgi secretion pathway in the pathogenic yeast Candida albicans, we cloned the C. albicans homologue of the Saccharomyces cerevisiae protein secretion gene YPT1. The C. albicans YPT1 ORF contained a 624 bp intronless ORF encoding a deduced protein of 207 aa and 23 kDa. This deduced protein was 77 % identical to S. cerevisiae Ypt1 protein (Ypt1p) and it contained GTP-binding domains that are conserved among all known ras-like GTPases. Multicopy plasmids containing C. albicans YPT1 complemented the temperature-sensitive S. cerevisiae ypt1 (A136D) mutation. One chromosomal YPT1 allele in C. albicans CAI4 was readily disrupted by homologous gene targeting, but attempts to disrupt the second allele yielded no viable null mutants. Since this suggested that C. albicans YPT1 may be essential, a mutant ypt1 allele was constructed encoding the amino acid substitution analogous to the N121I substitution in a known trans-dominant inhibitor of S. cerevisiae Ypt1p. Next, a GAL1-regulated plasmid was used to express the mutant ypt1(N121I) allele in C. albicans 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 membranebound secretory vesicles accumulated intracellularly. These results imply that C. albicans YPT1 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 C. albicans.

Functional Characterization of Myosin I Tail Regions in Candida albicans

“…The procedure for visualizing FM4-64 uptake was done as described by Vida and Emr (67). The secretion of secreted aspartyl proteases was monitored as described by Lee et al (35).…”

“…These results suggest that the IQ motifs and the TH1 region are required for myosin I function in fluid-phase endocytosis, whereas the TH2 region is only partially required. To determine if myosin I is also required for secretion in C. albicans, we examined the ability of the myosin I-deficient and wild-type strains to secrete aspartyl proteases (Saps) by using an assay that monitors the degradation of bovine serum albumin (BSA) in the medium (35). We found no significant difference between the wild type and the myosin I mutants ( Fig.…”

Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans

“…To test for secretory defects, liquid assays for secreted aspartyl proteases for strains THE1-CIp10 and tetR-SEC15 were performed as described previously (6,22). To induce production of Sap2p, cells from 5 ml overnight cultures were resuspended in 30 ml bovine serum albumin (BSA) medium (YNB without amino acids and without ammonium sulfate, 2% [wt/vol] glucose, 0.1% [wt/vol] BSA) and incubated for 24 h. Spent medium was removed, and the cells were resuspended to an OD 600 of 30 in fresh BSA medium containing only 0.01% (wt/vol) BSA.…”

Functional analysis ofCLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogenColletotrichum lindemuthianum

“…To study the role of these essential proteins, dominant-negative mutations were introduced into the wild-type sequence of their genes which were expressed under the control of an inducible promoter. This strategy provided a means to show that the C. albicans orthologues of yeast YPT1 and SEC4 are essential for growth and protease secretion (Lee et al, 2001;Mao et al, 1999).…”

“…In animal and plant pathogenic fungi, the role of these proteins in regulating protein transport is largely unknown. Recently, two Rab/GTPases from the human pathogen Candida albicans were analyzed (Lee et al, 2001;Mao et al, 1999). To study the role of these essential proteins, dominant-negative mutations were introduced into the wild-type sequence of their genes which were expressed under the control of an inducible promoter.…”