Ramabadran R S, Japa S, Beattie D S
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142, USA.
J Bioenerg Biomembr. 1997 Feb;29(1):45-54. doi: 10.1023/a:1022459722020.
The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc1 complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212-16218]. The deletion mutants lacking amino acid residues 55-66 or residues 161-180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161-180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc1 complex; however, the protein lacking residues 55-66 was assembled in vitro into the bc1 complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55-66. This deletion mutant protein was also assembled into the bc1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55-66, is not required for assembly of the iron-sulfur protein into the bc1 complex; however, this association did not lead to enzymatic activity of the bc1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.
将铁硫蛋白的两个缺失突变体体外导入从酵母菌株 JPJ1 分离的线粒体后，研究了它们组装到细胞色素 bc1 复合物中的情况，该酵母菌株的铁硫蛋白基因（RIP）已被删除。通过用针对铁硫蛋白或 bc1 复合物（复合物 III）的特异性抗血清从去污剂溶解的线粒体中免疫沉淀标记的铁硫蛋白或两个缺失突变体来研究组装过程[Fu 和 Beattie（1991）。《生物化学杂志》266，16212 - 16218]。缺少氨基酸残基 55 - 66 或残基 161 - 180 的缺失突变体在体外被导入线粒体，并通过中间形式加工成成熟形式。体外导入后，缺少残基 161 - 180 的蛋白质未组装到复合物中，这表明铁硫蛋白中包含这些残基的区域可能参与该蛋白质组装到 bc1 复合物中；然而，缺少残基 55 - 66 的蛋白质在体外与野生型铁硫蛋白一样有效地组装到 bc1 复合物中。此外，这种突变蛋白存在于从用含有该缺少残基 55 - 66 的蛋白质基因的单拷贝质粒转化的 JPJ1 细胞获得的线粒体膜部分中。这种缺失突变蛋白在体内也组装到 bc1 复合物中，这表明氨基酸的疏水片段，即残基 55 - 66，对于铁硫蛋白组装到 bc1 复合物中不是必需的；然而，这种结合并未导致 bc1 复合物的酶活性，因为在这些线粒体中 Rieske FeS 簇无法通过电子顺磁共振检测到。
