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首页> 外文期刊>Journal of bacteriology >Overexpression of a Dominant-Negative Allele ofSEC4 Inhibits Growth and Protein Secretion inCandida albicans
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Overexpression of a Dominant-Negative Allele ofSEC4 Inhibits Growth and Protein Secretion inCandida albicans

机译：SEC4的显性负等位基因的过表达抑制白色念珠菌的生长和蛋白质分泌。

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Candida albicans SEC4 was cloned by complementing theSaccharomyces cerevisiae sec4-8 mutation, and its deduced protein product (Sec4p) was 63% identical to S. cerevisiaeSec4p. 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.
机译：通过互补酿酒酵母sec4-8 突变克隆了白色念珠菌SEC4 ，其推导的蛋白质产物（Sec4p）与 S的同源性为63％。啤酒酵母 Sec4p。 C中有一个染色体 SEC4 等位基因。同源基因靶向很容易破坏白色念珠菌CAI4，但是努力破坏第二个等位基因没有产生可行的无效突变体。尽管这表明 C。白色念珠菌SEC4 是必不可少的，它没有提供有关该基因功能的信息。因此，我们构建了一个突变的 sec4 等位基因，其编码类似于 trans -主要抑制剂的Ser-17→Asn取代的氨基酸取代（Ser-28→Asn）。哺乳动物Ras蛋白。携带突变 sec4 等位基因（pS28N）的 GAL1 调控表达质粒在葡萄糖孵育的 C中影响最小。白色转化子，但测试的9个转化子中有6个在半乳糖中生长非常缓慢。 pS28N转化子在半乳糖中的孵育还抑制了天冬氨酰蛋白酶（Sap）的分泌，并导致90 nm分泌囊泡在细胞内积累，并且质粒固化将生长和Sap分泌恢复到野生型水平。这些结果表明 C。白色念珠菌SEC4 是生长和蛋白质分泌所必需的，它在蛋白质分泌途径中的作用比在高尔基体后分泌小泡的形成要晚。他们还证明了使用诱导型显性-阴性等位基因定义 C中必需基因的功能的可行性。白色的。

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