Inactivation of the gene encoding the 14‐kDa subunit VII of yeast ubiquinol

Abstract

The single nuclear gene encoding the 14‐kDa subunit VII of yeast ubiquinol:cytochrome c oxidoreductase has been inactivated by one‐step gene disruption, as verified by Southern blot analysis and immunoblotting. The resulting mutant has no ubiquinol:cytochrome c oxidoreductase activity and is respiratory‐deficient. Immunoblotting shows that cells lacking the 14‐kDa protein, also have lowered steady‐state levels of other subunits of complex III, the nuclear‐encoded 11‐kDa subunit VIII, the Rieske Fe‐S protein and the mitochondrially encoded cytochrome b can be detected spectrally. The steady‐state levels of the transcripts from genes encoding these proteins are not reduced, implying that the mutation exerts its pleiotropic effects at a posttranscriptional level. The residual amounts of subunits of complex III are recovered in the mutant mitochondria, suggesting that import is unaffected. The results strongly suggest that the 14‐kDa protein plays an essential role in the biosynthesis of the complex, most probably at the level of assembly. Field‐inversion gel electrophoresis was used to separate chromosomes of HR2 wild type and the (14‐kDa‐protein)° mutant, after which the gene encoding the 14‐kDa protein was located on chromosome IV by Southern blot analysis.

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

“…Results obtained earlier in our laboratory with a QCR7 disruption mutant in the wild-type strain HR2 showed that functional complementation was only possible after reintegration of the QCR7 gene into its original locus [20].…”

“…Western blot analysis on mitochondria1 protein isolated from 0.3% glucose, 2% galactose-grown cells of D11, D11-QCR7", and from null mutants transformed with either YCp-QCR7 or YEP-QCR7 showed that the Dll-QCR7" mutant has the same phenotype as described for the HR2-QCR7" mutant [20] and that, due to the absence of a complete QCR7 promoter [20], complementation of the QCR7" strain with the single copy plasmid YCp-QCR7 does not completely restore the levels of cytochrome 6, the Rieske FeS-protein and the 11-kDa subunit to the wild-type levels, while complementation with the multi-copy plasmid YEP-QCR7 does (data not shown, but see Fig. 5).…”

“…To be able to select for the URA3 gene, present on the plasmids YCplac33 and YEplacl95 [27], we decided to make a new QCR7 null mutant in the wild-type strain D11, using the knock-out construct described in [20].…”

“…The wild-type QCR7 gene was liberated as a 1.3-kb BarnHI-XbaI fragment, which does not contain the complete QCR7 promoter [20], from plasmid pEMBL9-14k (Fig. la) and cloned into the polylinker of the plasmids YCplac33 and YEplac 195 [24], digested with the same enzymes, giving plasmids YCp-QCR7 and YEP-QCR7 (Fig.…”

Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex

“…The petite phenotype resulting from deletion of subunit 9 differs from that which results from deletion of other nuclearencoded subunits, in that the subunit 9 deletion strain assembles an optically detectable cytochrome bc~ complex. In contrast, the loss of bc~ complex activity which results from deletion of core protein 1 [8], subunit 7 [9], or subunit 8 [10], can be attributed to lack of cytochrome b and/or incomplete assembly of the remaining subunits of the complex.…”