Crivellone M D, Wu M A, Tzagoloff A
Department of Biological Sciences, Columbia University, New York, New York 10027.
J Biol Chem. 1988 Oct 5;263(28):14323-33.
Coenzyme QH2-cytochrome c reductase is a multisubunit complex of the mitochondrial respiratory chain. Mutants of Saccharomyces cerevisiae with lesions in cytochromes b, c1, the non-heme iron protein, and the noncatalytic subunits have been used to study several aspects of the assembly of the complex. Strains with mutations in single subunits exhibit a variety of different phenotypes. Mutants in the 17-kDa (core 3) subunit grow normally on a nonfermentable substrate indicating that this component is not essential for either enzymatic activity or assembly of the enzyme. Mutations in all the other subunits express a respiratory-deficient phenotype and the absence of detectable enzyme activity. Among the respiratory-defective strains, some have mature cytochrome b (non-heme iron protein and cytochrome c1 mutants), while other mutants lack spectrally detectable cytochrome b and have reduced levels of the apoprotein (mutants in the 44-, 40-, 14-, and 11-kDa core subunits). Mutations in single subunits exert different effects on the concentrations of their partner proteins. These may be summarized as follows: 1) No substantial loss in the 44- or 40-kDa core subunits is seen in single mutants; 2) the concentration of cytochrome c1 is also relatively unaffected by mutations in the other subunits except for the cytochrome b mutant which has 60% of the wild type level of cytochrome c1; 3) all the single mutants have only 15-20% of the normal amount of non-heme iron protein; 4) mutations in the non-heme iron protein have no appreciable effect on the concentrations of the other subunits; 5) mutations in single subunits cause parallel decreases in the concentrations of cytochrome b, the 14-, and the 11-kDa subunits. These results indicate that the synthesis or stability of a subset of subunits depends on the presence of other subunit polypeptides of the complex. At present we favor the idea that the observed changes in the concentrations of some subunits are due to higher turnover rates of the proteins in a partially assembled complex. Based on the mutant phenotypes, a tentative model for the assembly of coenzyme QH2-cytochrome c reductase is proposed. According to this model it is envisioned that the subunits interact with one another in the lipid bilayer. Maturation of apocytochrome b occurs after it is assembled with the nonstructural subunits to form a core structure. This intermediate complex interacts with the non-heme iron protein to form the active holoenzyme.
辅酶QH2 - 细胞色素c还原酶是线粒体呼吸链的一个多亚基复合体。酿酒酵母中细胞色素b、c1、非血红素铁蛋白和非催化亚基发生损伤的突变体已被用于研究该复合体组装的几个方面。单个亚基发生突变的菌株表现出多种不同的表型。17 kDa（核心3）亚基发生突变的菌株在非发酵底物上能正常生长，这表明该组分对于酶活性或酶的组装并非必不可少。所有其他亚基发生突变的菌株均表现出呼吸缺陷表型且缺乏可检测到的酶活性。在呼吸缺陷型菌株中，一些菌株具有成熟的细胞色素b（非血红素铁蛋白和细胞色素c1突变体），而其他突变体缺乏光谱可检测的细胞色素b且脱辅基蛋白水平降低（44 kDa、40 kDa、14 kDa和11 kDa核心亚基的突变体）。单个亚基发生突变对其伴侣蛋白的浓度产生不同影响。这些影响可总结如下：1）在单个突变体中未观察到44 kDa或40 kDa核心亚基有大量损失；2）细胞色素c1的浓度相对不受其他亚基突变的影响，但细胞色素b突变体的细胞色素c1水平为野生型的60%；3）所有单个突变体的非血红素铁蛋白含量仅为正常量的15% - 20%；4）非血红素铁蛋白发生突变对其他亚基的浓度没有明显影响；5）单个亚基发生突变会导致细胞色素b、14 kDa和11 kDa亚基的浓度平行下降。这些结果表明，一部分亚基的合成或稳定性取决于该复合体中其他亚基多肽的存在。目前我们倾向于认为，观察到的一些亚基浓度变化是由于部分组装的复合体中蛋白质的更新率较高所致。基于突变体表型，提出了辅酶QH2 - 细胞色素c还原酶组装的一个初步模型。根据该模型，设想亚基在脂质双层中相互作用。脱辅基细胞色素b与非结构亚基组装形成核心结构后发生成熟。这个中间复合体与非血红素铁蛋白相互作用形成有活性的全酶。
