Malaney S, Trumpower B L, Deber C M, Robinson B H
Department of Genetics, the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 1997 Jul 11;272(28):17495-501. doi: 10.1074/jbc.272.28.17495.
Subunit 7 of the yeast cytochrome bc1 complex is encoded by the nuclear QCR7 gene and is essential for respiration. This protein does not contain a cleavable N-terminal mitochondrial targeting sequence, and it is not understood how the Qcr7 protein is imported into mitochondria and assembled into the complex. To test the role of the N terminus of the Qcr7 protein in mitochondrial import, assembly of the complex, and proton translocation, we inactivated the endogenous QCR7 gene and expressed mutated qcr7 genes capable of synthesizing proteins truncated by 7, 10, 14, and 20 residues (Qcr7p-delta7, Qcr7p-delta10, Qcr7p-delta14, and Qcr7p-delta20, respectively) from the N terminus. In addition, we studied two mutants containing Qcr7 proteins with point mutations in addition to a delta7 truncation, Qcr7p-delta7(D13V) and Qcr7p-delta7(R10K). All the mutant proteins with the exception of Qcr7p-delta10 were present in the mitochondria at 30 degrees C, although most at lower steady-state levels than the Qcr7p from the strain overexpressing wild type QCR7. The absence of the Qcr7p-delta10 may be the result of an unstable protein or a decrease in the efficiency of mitochondrial import due to its compromised amphipathic alpha-helix and the presence of a negative charge exposed at the N terminus. Cytochrome c reductase activities and the amounts of ATP synthesized were comparable with the wild type in the strain expressing Qcr7p-delta7. The strain expressing Qcr7p-delta7(R10K) had an identical phenotype to the one containing the Qcr7p-delta7, whereas strains expressing the Qcr7p-delta10, Qcr7p-delta14, Qcr7p-delta20, and Qcr7p-delta7(D13V) were all respiration-deficient. Examination of the steady-state levels of complex III subunits showed that core protein 2, cytochrome c1, the iron-sulfur protein, and the 11-kDa subunit are reduced in respiration-deficient mutant strains. Results from deletion analyses indicate that the N-terminal 20 residues (after Met-1) of the Qcr7 protein are not essential for import into mitochondria and that the N-terminal seven residues (after Met-1) are not involved in proton translocation. The results of this work show, however, that the N terminus of the Qcr7 protein is essential for the biosynthesis of ubiquinol-cytochrome c reductase.
酵母细胞色素bc1复合物的亚基7由核基因QCR7编码，对呼吸作用至关重要。该蛋白不包含可切割的N端线粒体靶向序列，目前尚不清楚Qcr7蛋白是如何导入线粒体并组装成复合物的。为了测试Qcr7蛋白N端在线粒体导入、复合物组装和质子转运中的作用，我们使内源性QCR7基因失活，并表达了能够合成从N端截短7、10、14和20个残基的蛋白质的突变qcr7基因（分别为Qcr7p-Δ7、Qcr7p-Δ10、Qcr7p-Δ14和Qcr7p-Δ20）。此外，我们研究了两个除了Δ7截短外还含有Qcr7蛋白点突变的突变体，即Qcr7p-Δ7(D13V)和Qcr7p-Δ7(R10K)。除了Qcr7p-Δ10外，所有突变蛋白在30℃时都存在于线粒体中，尽管大多数的稳态水平低于过表达野生型QCR7菌株的Qcr7p。Qcr7p-Δ10的缺失可能是由于蛋白质不稳定，或者由于其两亲性α-螺旋受损以及N端暴露的负电荷导致线粒体导入效率降低。在表达Qcr7p-Δ7的菌株中，细胞色素c还原酶活性和合成的ATP量与野生型相当。表达Qcr7p-Δ7(R10K)的菌株与含有Qcr7p-Δ7的菌株具有相同的表型，而表达Qcr7p-Δ10、Qcr7p-Δ14、Qcr7p-Δ20和Qcr7p-Δ7(D13V)的菌株均存在呼吸缺陷。对复合物III亚基稳态水平的检测表明，在呼吸缺陷突变菌株中，核心蛋白2、细胞色素c1、铁硫蛋白和11 kDa亚基的水平降低。缺失分析结果表明，Qcr7蛋白的N端20个残基（Met-1之后）对于导入线粒体不是必需的，N端7个残基（Met-1之后）不参与质子转运。然而，这项工作的结果表明，Qcr7蛋白的N端对于泛醌-细胞色素c还原酶的生物合成是必需的。
