Yeast ubiquinol: cytochrome c oxidoreductase is still active after inactivation of the gene encoding the 17‐kDa subunit VI

Abstract

The single nuclear gene encoding the 17-kDa subunit VI of yeast ubiquinol: cytochrome c oxidoreductase has been inactivated by one-step gene disruption. Disruption was verified by Southern blot analysis of nuclear DNA and immunoblotting. Cells lacking the 17-kDa protein are still capable of growth on glycerol and they contain all other subunits of complex 111 at wild-type levels, implying that the 17-kDa subunit is not essential for either assembly of complex 111, or its function. In vitro, electron transport activity of complex I11 of mutant cells is about 40% of the wild-type complex, but for the total respiratory chain no significant difference in activity was measured between mutant and wild type. The energy-transducing capacity of the complex is not reduced in the absence of the 17-kDa protein. In a relatively high proportion of the transformants, disruption of the 17-kDa gene was accompanied by the appearance of a second mutation causing a petite phenotype. In these cells which lack cytochrome b, the presence of the 17-kDa protein (after complementation) results in stabilization of cytochrome c l .The respiratory chain enzyme ubiquinol : cytochrome c oxidoreductase in yeast is thought to consist of 8 -10 different subunits [l, 21. The enzyme catalyzes electron transfer from ubiquinol to cytochrome c. Coupled to this process is the translocation of protons over the mitochondria1 inner membrane, resulting in an electrochemical gradient which is used by the FoF1-ATPase to generate ATP.Three subunits, cytochrome b, cytochrome c1 and the Rieske Fe-S protein, with molecular masses of 42, 29 and 24 kDa respectively, contain a prosthetic group and play an essential role in electron transfer. The remaining subunits are the two core proteins, I and 11, of 44 and 40 kDa respectively, three smaller polypeptides of 17, 14 and 11 kDa and possibly two proteins of 8 and 5.5 kDa [l, 21. Until recently, nothing was known about a possible function of these proteins in electron transfer, proton translocation and/or assembly of the complex. Since then, however, genes coding for most of these subunits have been cloned [3 -51 and mutants, in which the gene coding for each has been inactivated, have been constructed [5-71. Analysis of these mutants shows that the 11-kDa, 14-kDa and 44-kDa proteins are essential: their absence results in lack of assembly and proteolytic degradation of several other subunits [5, 61. Absence of the 40-kDa core I1 protein also destabilizes the remaining complex but in this mutant approximately 5% of the wild-type complex 111 activity remains, indicating that this protein is not essential for electron transfer [7].In the present study we report on the construction and characterization of a mutant lacking the 17-kDa subunit VI.

UQCRH gene encoding mitochondrial Hinge protein is interrupted by a translocation in a soft-tissue sarcoma and epigenetically inactivated in some cancer cell lines

“…The crystal structure of complex III has been resolved (Iwata et al, 1998), providing further evidence that the peculiar feature of nine consecutive glutamic residues at the N-terminal of the Hinge protein may be the site of interaction with the basic protein cytochrome c. The protein is highly conserved from yeast to humans. In yeast, the 17 kDa subunit of complex III is the homolog of Hinge protein and deletion mutants are still capable of growth on a nonfermentable substrate, therefore implying that it is not essential for the assembly and function of complex III (Crivellone et al, 1988;Schoppink et al, 1988). Nonetheless, there is evidence that it may exert a regulatory role of complex III and of its association with cytochrome c (Kim et al, 1989;Schoppink et al, 1989).…”

The C‐terminus of the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme

“…Mitochondria were isolated as described earlier [29]. Mitochondria1 proteins were separated on SDS/polyacrylamide slab gels according to [30] and blotted according to [31].…”

Membrane topography of the subunits of ubiquinol – cytochrome‐c oxidoreductase of Saccharomyces cerevisiae

“…Isolation was performed according to published methods [16]. Prior to the digitonin titrations, mitochondria were diluted to 5 mg proteinjml in 0.6 M sorbitol, 25 mM potassium phosphate pH 7.4.…”