Wu M, Tzagoloff A
Department of Biological Sciences, Columbia University, New York, New York 10027.
J Biol Chem. 1989 Jul 5;264(19):11122-30.
Respiratory defective mutants of Saccharomyces cerevisiae assigned to complementation group G28 display a deficiency, in the respiratory chain complex coenzyme QH2-cytochrome c reductase. The mutants define a new nuclear gene, designated CBP3, required for the assembly of the complex. Mutations in CBP3 are expressed in the absence of spectrally and immunologically detectable cytochrome b, a catalytic subunit of coenzyme QH2-cytochrome c reductase. The mutational block responsible for the cytochrome b deficiency has been ascribed to a post-translational step based on the observation that cbp3 mutants have wild type concentrations of cytochrome b mRNA and are capable of synthesizing the apoprotein. Western analysis has revealed that cbp3 mutants have reduced levels of a subset of subunit polypeptides of the coenzyme QH2-cytochrome c reductase complex that include apocytochrome b, the iron-sulfur protein, core 4 (14-kDa subunit), and core 5 (11-kDa subunit). A similar phenotype has previously been reported in strains that fail to assemble the complex as a result of mutations in the noncatalytic core subunits. The CBP3 gene has been cloned by transformation of a mutant from complementation group G28 with a yeast genomic library. The gene is 1005 nucleotides long and codes for a primary translation product of 39 kDa. A transcript of a size commensurate with the length of the CBP3 reading frame is detected in total and poly(A+)-enriched RNA. The amino-terminal region of the CBP3 product is basic and probably corresponds to a cleavable mitochondrial targeting signal. An antibody obtained against a trpE/CBP3 fusion protein detects a protein of 40 kDa in wild type yeast mitochondria. This protein is absent in a mutant construct containing a partially deleted copy of the gene. The CBP3 protein is a membrane constituent, although attempts to demonstrate its physical association with the other subunits of coenzyme QH2-cytochrome c reductase have been unsuccessful.
酿酒酵母呼吸缺陷型突变体被归为互补群G28，其在呼吸链复合物辅酶QH2 - 细胞色素c还原酶中表现出缺陷。这些突变体定义了一个新的核基因，命名为CBP3，它是该复合物组装所必需的。CBP3中的突变在缺乏光谱和免疫可检测的细胞色素b（辅酶QH2 - 细胞色素c还原酶的催化亚基）的情况下表达。基于cbp3突变体具有野生型浓度的细胞色素b mRNA且能够合成脱辅基蛋白这一观察结果，导致细胞色素b缺乏的突变阻断已归因于翻译后步骤。蛋白质免疫印迹分析表明，cbp3突变体中辅酶QH2 - 细胞色素c还原酶复合物的亚基多肽子集水平降低，这些亚基包括脱辅基细胞色素b、铁硫蛋白、核心4（14 kDa亚基）和核心5（11 kDa亚基）。先前在因非催化核心亚基突变而未能组装该复合物的菌株中也报道过类似的表型。通过用酵母基因组文库转化互补群G28的一个突变体，克隆了CBP3基因。该基因长1005个核苷酸，编码一个39 kDa的初级翻译产物。在总RNA和富含多聚腺苷酸（poly(A+)）的RNA中检测到与CBP3阅读框长度相当的转录本。CBP3产物的氨基末端区域呈碱性，可能对应于一个可裂解的线粒体靶向信号。针对trpE/CBP3融合蛋白获得的抗体在野生型酵母线粒体中检测到一个40 kDa的蛋白质。在含有该基因部分缺失拷贝的突变构建体中不存在这种蛋白质。CBP3蛋白是一种膜成分，尽管试图证明它与辅酶QH2 - 细胞色素c还原酶的其他亚基存在物理关联但未成功。
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