The N Terminus of the Qcr7 Protein of the Cytochromebc 1 Complex Is Not Essential for Import into Mitochondria in Saccharomyces cerevisiae but Is Essential for Assembly of the Complex

Abstract

Subunit 7 of the yeast cytochrome bc 1 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-⌬7, Qcr7p-⌬10, Qcr7p-⌬14, and Qcr7p-⌬20, respectively) from the N terminus. In addition, we studied two mutants containing Qcr7 proteins with point mutations in addition to a ⌬7 truncation, Qcr7p-⌬7(D13V) and Qcr7p-⌬7(R10K). All the mutant proteins with the exception of Qcr7p-⌬10 were present in the mitochondria at 30°C, although most at lower steady-state levels than the Qcr7p from the strain overexpressing wild type QCR7. The absence of the Qcr7p-⌬10 may be the result of an unstable protein or a decrease in the efficiency of mitochondrial import due to its compromised amphipathic ␣-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-⌬7. The strain expressing Qcr7p-⌬7(R10K) had an identical phenotype to the one containing the Qcr7p-⌬7, whereas strains expressing the Qcr7p-⌬10, Qcr7p-⌬14, Qcr7p-⌬20, and Qcr7p-⌬7(D13V) were all respiration-deficient. Examination of the steady-state levels of complex III subunits showed that core protein 2, cytochrome c 1 , 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.

Further insights into the assembly of the yeast cytochrome bc1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b

“…The results with the deletion mutant lacking subunit 7 further corroborate the interdependence among subunits 7, 8 and cytochrome b and the role of this core subcomplex in organizing the cytochrome bc 1 complex. It was proposed previously that the N‐terminus of subunit 7 plays an important role during the assembly of the cytochrome bc 1 complex [33,34]. In support of this proposal, it is the N‐terminal 30 amino acids of subunit 7 that envelopes the N‐terminus of cytochrome b near the matrix side of the inner membrane (Fig.…”

A Defect in Coenzyme Q Biosynthesis Is Responsible for the Respiratory Deficiency in Saccharomyces cerevisiae abc1Mutants

“…Cytochrome c oxidase activities were assayed at 30°C in 40 mM sodium phosphate buffer, pH 7.4, containing 25 M reduced cytochrome c (43,44). Horse heart cytochrome c was reduced in 40 mM sodium phosphate buffer, pH 7.4, with L-ascorbate, and the ascorbate was subsequently removed by dialysis.…”

“…Horse heart cytochrome c was reduced in 40 mM sodium phosphate buffer, pH 7.4, with L-ascorbate, and the ascorbate was subsequently removed by dialysis. The reaction was initiated by adding 25 g of mitochondrial protein and the reaction rate was monitored by following the oxidation of cytochrome c at 550 Ϫ 540 nm for 30 s (44). Cytochrome c oxidase activities were determined from the initial linear rate of reaction using an extinction coefficient of 18.5 mM Ϫ1 cm Ϫ1 (43).…”

Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA

“…The red colouration depends on active respiration and in the absence of respiration white colonies are formed. This colour phenotype allowed for a rapid assay of mitochondrial function on rich media (Greenleaf et al , 1986; Malaney et al , 1997). Since the heterozygous diploid grew on YPEG media and developed the red colouration indicative of active respiration, one copy of the wild‐type allele of HMI1 is sufficient to ensure the presence of functional mitochondria.…”