Inactivation of the gene encoding the 14-kDa subunit VII of yeast ubiquinol. Cytochrome c oxidoreductase and analysis of the resulting mutant.

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. No 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 post-transcriptional 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) degrees mutant, after which the gene encoding the 14-kDa protein was located on chromosome IV by Southern blot analysis.

The C-terminal half of the 11-kDa subunit VIII is not necessary for the enzymic activity of yeast ubiquinol:cytochrome-c oxidoreductase.

Inactivation of the gene encoding the 11-kDa subunit VIII of yeast ubiquinol:cytochrome c oxidoreductase leads to an inactive complex, which lacks detectable cytochrome b [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A. and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184] and in which the steady-state levels of the Fe-S protein and the 14-kDa subunit VII are severely reduced. When the 11-kDao mutant is transformed with a gene encoding a protein consisting of the 11-kDa protein minus its last 11 amino acids and fused to a 7-amino-acid sequence encoded by a stop oligonucleotide, the complex is assembled normally. Enzyme activity is similar to that of the wild type, as is also the sensitivity of the complex to antimycin and myxothiazol. Transformation of the mutant with a gene encoding a protein consisting of the 11-kDa protein lacking the last 43 amino acids (i.e. almost half the protein) and fused to the same 7-amino-acid sequence as above, gives partial restoration of the complex. The Fe-S protein and the 14-kDa subunit VII still exhibit low steady-state levels, but cytochrome b is present again, albeit at a strongly reduced level. Electron transport activity is also partially restored and correlates with the level of cytochrome b indicating that the turnover number of the complex is similar to that of wild-type complex III. These findings demonstrate the important role played by the 11-kDa protein in the stabilization of cytochrome b. They also imply that at least the C-terminal half of the 11-kDa protein is not part of an ubiquinol-binding site. Moreover, since the deletion has no effect on the sensitivity of the complex to myxothiazol and antimycin, at least this part of the protein is probably not involved in binding of these inhibitors.

The effect of deletion of the genes encoding the 40 kDa subunit II or the 17 kDa subunit VI on the steady-state kinetics of yeast ubiquinol-cytochrome-c oxidoreductase.

Yeast ubiquinol-cytochrome c oxidoreductase is still active after inactivation of the genes encoding the 40 kDa Core II protein or the 17 kDa subunit VI (Oudshoorn et al. (1987) Eur. J. Biochem. 163, 97-103 and Schoppink et al. (1988) Eur. J. Biochem. 173, 115-122). The steady-state levels of several other subunits of Complex III are severely reduced in the 40 kDa0 mutant. The level of spectrally detectable Complex III cytochrome b in the mutant submitochondrial particles is about 5% of that of the wild type. However, when the steady-state activity of Complex III with respect to the cytochrome c reduction was examined, similar maximal turnover numbers and Km values were found for the mutated and the wild-type complexes, both when yeast cytochrome c and when horse-heart cytochrome c was used as electron acceptor. We therefore conclude that the Core II subunit of yeast Complex III plays no role in the binding of cytochrome c and that it has no major influence of the overall electron transport and on the binding of ubiquinol by the enzyme. Absence of the 17 kDa subunit VI of yeast Complex III, the homologous counterpart of the hinge protein of the bovine heart enzyme, resulted in a decrease in the rate of reduction of both horse-heart cytochrome c and yeast cytochrome c by Complex III under conditions of relatively high ionic strength. However, under conditions of optimal ionic strength, no difference could be seen in the maximal turnover numbers and Km values, neither with horse-heart cytochrome c nor with yeast cytochrome c between Complex III deficient in the 17 kDa protein and the wild-type complex. Binding of ATP to ferricytochrome c inhibits its reduction by Complex III under conditions of relatively high ionic strength. But when the 17 kDa protein is absent, this inhibition is also observed under optimal ionic-strength conditions. These results can be explained by assuming a stimulating role for the acidic 17 kDa protein in the association of basic cytochrome c with Complex III. This association is (part of) the rate-limiting step in the reduction of cytochrome c by Complex III under conditions of relatively high ionic strength or when this association is hindered, for instance, by binding of ATP.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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 III at wild-type levels, implying that the 17-kDa subunit is not essential for either assembly of complex III, or its function. In vitro, electron transport activity of complex III of mutant cells is about 40% of the wild-type complex, but for the total respiratory chain no significant differences 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 c1.

Inactivation of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae.

The single nuclear gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase (complex III) in Saccharomyces cerevisiae has been inactivated by a one-step gene disruption procedure. Inactivation results in a loss of ubiquinol-cytochrome-c oxidoreductase activity (less than 1% wild type) and respiratory deficiency. Cells lacking the 11-kDa protein also display lowered steady-state levels of other complex-III subunits encoded by nuclear genes including the 14-kDa subunit VII and the Rieske Fe-S protein and of the mitochondrially encoded cytochrome b. The steady-state levels of the transcripts from the genes encoding these proteins are however not reduced. The results strongly imply that the 11-kDa protein plays an important role in regulating the synthesis of complex III at the post-transcriptional level, most likely assembly. Separation of chromosomes by pulsed-field gel electrophoresis of DNA of wild-type and of the mutant lacking the 11-kDa-protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XII as reported by Van Loon et al. [Mol. Gen. Genet. 197 (1984) 219-224].

Yeast ribosomal protein S33 is encoded by an unsplit gene.

The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis. The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD. Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed. By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.