Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7235-7242 |
| Number of pages | 8 |
| Journal | Journal of Bacteriology |
| Volume | 181 |
| Issue number | 23 |
| State | Published - Dec 1999 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Immunology
Cite this
Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans. / Mao, Yuxin; Kalb, Vernon F.; Wong, Brian.
In: Journal of Bacteriology, Vol. 181, No. 23, 12.1999, p. 7235-7242.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans
AU - Mao, Yuxin
AU - Kalb, Vernon F.
AU - Wong, Brian
PY - 1999/12
Y1 - 1999/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0032698991&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032698991&partnerID=8YFLogxK
M3 - Article
VL - 181
SP - 7235
EP - 7242
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 23
ER -