The N terminus of the Qcr7 protein of the cytochrome bc1 complex is not essential for import into mitochondria in Saccharomyces cerevisiae but is essential for assembly of the complex.
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.
Malaney
S
S
Department of Genetics, the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Trumpower
B L
BL
Deber
C M
CM
Robinson
B H
BH
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010101:Oxygen Consumption; D011487:Protein Conformation; D017494:Proton Pumps / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D013696:Temperature
D046911:Macromolecular Substances; D017494:Proton Pumps; D014450:Electron Transport Complex III