The function of the Qcr7 protein of the mitochondrial ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae

The respiratory chain enzyme ubiquinol-cytochrome c oxidoreductase (also termed $bc\sb1$ complex or complex III) of Saccharomyces cerevisiae contains 10 subunits and resides in the inner mitochondrial membrane (IMM). This multisubunit enzyme complex is involved in the transfer of electrons from ubiquinol to cytochrome c and in the establishment of an electrochemical gradient by translocation of protons across the IMM. A previous study has shown that inactivation of the yeast nuclear gene QCR7, which encodes subunit 7 (also referred to as Qcr7 protein or 14 kDa subunit) of the $bc\sb1$ complex leads to an inactive enzyme. The $bc\sb1$ complex of the mutant strain lacks holocytochrome b and has reduced levels of apocytochrome b, the Rieske iron-sulfur protein (ISP), and the 11 kDa subunit. Although this study showed that the Qcr7 protein is essential for respiration, the exact role of this subunit is not known. In the present study I have shown by circular dichroism that an amino-terminal peptide of subunit 7 is $\alpha$-helical. I have also studied the effect of mutations in the amino-terminus of this protein on mitochondrial targeting, assembly of the $bc\sb1$ complex, and proton translocation. Mutant proteins were analyzed by overexpression of mutated qcr7 genes in the strain YSM-qcr7$\Delta$, which contains a disrupted chromosomal QCR7 gene, and by comparison to Qcr7 protein from a YSM-qcr7$\Delta$ strain in which the wild type QCR7 gene is overexpressed. Respiration proficiency and the activity of the $bc\sb1$ complex were monitored. Based on these preliminary data, strains expressing mutated proteins that lacked the N-terminal 7, 10, 14, and 20 residues (after Met-1) of subunit 7 referred to as Qcr7p-$\Delta$7, -$\Delta$10, -$\Delta$14, and -$\Delta$20, respectively, and strains expressing versions of Qcr7p-$\Delta$7 that contained point mutations R10K, D13V, and R10I/G12V were chosen for further study. All the mutated versions of the Qcr7 protein, with the exception of Qcr7p-$\Delta$10 and Qcr7p-$\Delta$7(D13V), are present in the mitochondria (from cells grown at 30$\sp\circ$C) at reduced levels of approximately 55% when compared to Qcr7 protein from the strain overexpressing wild type QCR7. In contrast, Qcr7p-$\Delta$10 is not detectable and Qcr7p-$\Delta$7(D13V) is present at wild type levels. This may implicate the Qcr7 protein amino-terminus in import or in conferring stability to the protein. The activity of complex III in mitochondria from strains with Qcr7p-$\Delta$7 and Qcr7p-$\Delta$7(R10K) was normal. In contrast, strains overexpressing qcr7 genes encoding Qcr7p-$\Delta$10, Qcr7p-$\Delta$14, Qcr7p-$\Delta$20, Qcr7p-$\Delta$7(D13V), and Qcr7p-$\Delta$7(R10I/G12V) were respiration-deficient. Western blot analyses from this latter group of mutants indicate that the $bc\sb1$ complex in respiration-deficient cells displays a significant variation of reduced levels of the 11kDa subunit, as well as combined intermediate and mature ISP and cytochrome $c\sb1$. Spectrophotometric analyses indicate that the amount of holocytochrome b was reduced in the strain containing Qcr7p-$\Delta$7(R10I/G12V) and lacking in the strain with Qcr7p-$\Delta$7(D13V). ATP synthesis (an indirect measure of proton translocation) in mitochondria was comparable to the wild type in all respiration-proficient strains tested, including the strain with Qcr7p-$\Delta$7. Based on the results of this work, I concluded that the amino-terminus of the Qcr7 protein is essential for the functional assembly of ubiquinol-cytochrome c oxidoreductase. In addition to the proposed function in assembly, the N-terminal seven residues may facilitate import into mitochondria.