Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle
Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle
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DOI：
10.1074/jbc.272.52.33191
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发表时间：
1997-12-26
影响因子：
4.8
通讯作者：
Tzagoloff, A
中科院分区：
文献类型：
--
作者：
Beers, J;Glerum, DM;Tzagoloff, A
Cox17p是细胞色素氧化酶在酿酒酵母中表达所必需的。在本研究中，COX17被置于GAL10启动子的控制下，该启动子位于一个自主复制的质粒中。用含有高拷贝结构的酵母转化子纯化Cox17p，使其纯化到均一。纯化的Cox17p每摩尔蛋白质含有0.2-0.3摩尔的铜。在还原条件下，在6倍摩尔过量的氯化亚铜存在下，Cox17P孵化后，铜的摩尔含量增加到1.8。用牛血清白蛋白偶联的羧基末端多肽免疫兔，获得了抗Cox17p的抗体。该抗血清在野生型酵母和高表达Cox17p的转化子的线粒体和可溶性蛋白组分中均检测到Cox17p。将完整的线粒体暴露在低张条件下，导致Cox17p的大部分以可溶性蛋白质的形式释放，表明Cox17p的线粒体部分定位于膜间隙。这些结果与先前提出的Cox17p的功能一致，即为线粒体利用提供细胞质铜。
Cox17p was previously shown to be essential for the expression of cytochrome oxidase in Saccharomyces cerevisiae. In the present study COX17 has been placed under the control of the GAL10 promoter in an autonomously replicating plasmid. A yeast transformant harboring the high copy construct was used to purify Cox17p to homogeneity. Purified Cox17p contains 0.2-0.3 mol of copper per mol of protein. The molar copper content is increased to 1.8 after incubation of Cox17p in the presence of a 6-fold molar excess of cuprous chloride under reduced conditions. An antibody against Cox17p was obtained by immunization of rabbits with a carboxyl-terminal peptide coupled to bovine serum albumin. The antiserum detects Cox17p in both the mitochondrial and soluble protein fractions of wild type yeast and of the transformant overexpressing Cox17p. Exposure of intact mitochondria to hypotonic conditions causes most of Cox17p to be released as a soluble protein indicating that the mitochondrial fraction of Cox17p is localized in the intermembrane space. These results are consistent with the previously proposed function of Cox17p, namely in providing cytoplasmic copper for mitochondrial utilization.