Robinson K M, Lemire B D
Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1996 Feb 23;271(8):4061-7. doi: 10.1074/jbc.271.8.4061.
Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial heterotetramer containing a flavoprotein subunit with an 8alpha-N(3)-histidyl-linked FAD cofactor. The covalent linkage of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein precursor in mitochondrial matrix fractions. Flavoprotein modification does not depend on translocation across a membrane, but it does require proteolytic processing by the mitochondrial processing peptidase prior to flavin attachment. Since ATP depletion, N-ethylmaleimide, or proteinase treatments of matrix fractions inhibit flavoprotein modification, at least one additional matrix protein component appears to be required. Having previously suggested that the flavoprotein begins folding before FAD attachment occurs, we tested whether the mitochondrial chaperonin, heat shock protein 60, might be necessary. Co-immunoprecipitation of the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matrix fractions does not inhibit FAD attachment. Nonprotein components are also required for flavoprotein modification. In addition to ATP, effector molecules such as succinate, fumarate, or malate also stimulate modification. Together, these results suggest that FAD addition is an early event in succinate dehydrogenase assembly.
酿酒酵母中的琥珀酸脱氢酶（EC 1.3.99.1）是一种线粒体异源四聚体，包含一个黄素蛋白亚基，该亚基带有一个8α-N(3)-组氨酰连接的FAD辅因子。FAD的共价连接对于酶活性是必需的。我们开发了一种体外测定法，用于测量线粒体基质组分中黄素蛋白前体的黄素化。黄素蛋白的修饰不依赖于跨膜转运，但在黄素附着之前确实需要线粒体加工肽酶进行蛋白水解加工。由于对基质组分进行ATP耗竭、N-乙基马来酰亚胺或蛋白酶处理会抑制黄素蛋白的修饰，因此似乎至少还需要一种额外的基质蛋白成分。此前我们曾提出黄素蛋白在FAD附着之前就开始折叠，我们测试了线粒体伴侣蛋白热休克蛋白60是否可能是必需的。黄素蛋白和伴侣蛋白的共免疫沉淀表明这两种蛋白确实相互作用。然而，从基质组分中免疫去除伴侣蛋白并不抑制FAD的附着。黄素蛋白修饰还需要非蛋白质成分。除了ATP外，琥珀酸、富马酸或苹果酸等效应分子也会刺激修饰。这些结果共同表明，FAD添加是琥珀酸脱氢酶组装过程中的一个早期事件。
