Lawson J E, Behal R H, Reed L J
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Biochemistry. 1991 Mar 19;30(11):2834-9. doi: 10.1021/bi00225a015.
Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)
在酿酒酵母中，编码线粒体丙酮酸脱氢酶（PDH）复合体X蛋白组分的PDX1基因的破坏并不影响细胞的活力。然而，与野生型线粒体提取物相比，突变体的线粒体提取物不能催化丙酮酸的辅酶A和NAD⁺连接的氧化反应。从突变细胞中分离出的PDH复合体含有丙酮酸脱氢酶（E1α + E1β）和二氢硫辛酰胺乙酰转移酶（E2），但缺少X蛋白和二氢硫辛酰胺脱氢酶（E3）。用单位拷贝质粒上的X蛋白基因转化的突变细胞产生了一种PDH复合体，该复合体含有X蛋白和E3，以及E1α、E1β和E2，并且表现出与野生型PDH复合体相似的总体活性。这些观察结果表明，X蛋白不参与E2核心的组装，也不是E2核心的组成部分。相反，X蛋白显然在PDH复合体中起结构作用；即，它将E3结合并定位到E2核心上，这种特异性结合对于功能性PDH复合体至关重要。通过缺失突变获得了支持该结论的额外证据。缺失X蛋白大部分硫辛酰结构域（第6 - 80位氨基酸残基）对PDH复合体的总体活性影响很小。这一观察结果表明，硫辛酰结构域及其共价结合的硫辛酰部分对于X蛋白的功能并非必不可少。然而，缺失X蛋白假定的亚基结合结构域（约第144 - 180位氨基酸残基）导致E3的高亲和力结合丧失，同时PDH复合体的总体活性也丧失。（摘要截短于250字）
