Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans
Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans
复制标题
DOI：
10.1128/jb.181.23.7235-7242.1999
复制
发表时间：
1999-12-01
影响因子：
3.2
通讯作者：
Wong, B
中科院分区：
文献类型：
--
作者：
Mao, YX;Kalb, VF;Wong, B
通过对酿酒酵母（Saccharomycescerevisiae）的sec 4 -8突变进行互补，克隆了白色念珠菌（Candida albicans）的SEC 4基因，其推导的蛋白产物（Sec 4p）与S.酿酒酵母Sec 4p.白色念珠菌CAI 4中的一个染色体SEC 4等位基因很容易被同源基因靶向破坏，但破坏第二个等位基因的努力没有产生可行的无效突变体。尽管这表明C.白色念珠菌SEC 4基因是必需的，但它没有提供有关该基因功能的信息。因此，我们构建了一个编码类似于哺乳动物Ras蛋白反式显性抑制剂中Ser-17-->Asn替换的氨基酸替换（Ser-28-->Asn）的突变体sec 4等位基因，携带突变体sec 4等位基因（pS28 N）的GAL 1调控的表达质粒在葡萄糖孵育的C. albicans转化体，但测试的9个转化体中的6个在半乳糖中生长非常缓慢。在半乳糖中孵育pS28 N转化体也抑制了乙酰基蛋白酶（Sap）的分泌，并导致90-nm分泌囊泡在细胞内积累，质粒固化恢复了生长和Sap分泌至野生型水平。白色念珠菌SEC 4是生长和蛋白质分泌所必需的，并且它在蛋白质分泌途径中比形成后高尔基体分泌囊泡。它们也证明了利用可诱导的显性负等位基因来确定C.白色念珠菌
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.