Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes

Loon, A. P. G. M. Van; Vijn, R. J.; Groot, Raoul J. de; Polman, J. E. M.; Grivell, Leslie A.

doi:10.1007/bf00330966

Cited by 9 publications

(3 citation statements)

References 20 publications

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“…One of the former class of transformants was selected for further study. Southern blot hybridization (data not shown) indicates that integration of transforming DNA has indeed taken place within the single gene for subunit I1 [33]. As shown in Table 1, cells display strongly reduced, but measurable levels of ubiquinol : cytochrome-c oxidoreductase activity (5% relative to wild type; about 50% of this activity is sensitive to antimycin A and myxothiazol).…”

Section: Gene Disruptionmentioning

confidence: 94%

Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

Oudshoorn

Steeg

Swinkels

et al. 1987

European Journal of Biochemistry

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The nucleotide sequence of a 2.5 x 103-base segment of yeast nuclear DNA, containing the structural gene for the 40-kDa subunit I1 of the ubiquinol: cytochrome-c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit 11. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40-kDa protein shows that subunit I1 is synthesized as a longer precursor and shows that the extension is N-terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA . RNA hybrids identifies two major and one minor transcript of the gene, whose 5' termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5' of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit I1 gene has been constructed by a one-step gene-disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH,: cytochrome-c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit I1 in the complex are discussed.QH, : cytochrome-c oxidoreductase is a proton-translocating enzyme of the respiratory chain in mitochondria. In yeast it consists of eight to ten subunits [l -31, of which only one, apocytochrome b, is mitochondrially encoded [4, 51. The remaining subunits are encoded in the nucleus. The dual genetic origin of the enzyme poses questions concering the coordinate synthesis, import and assembly of individual subunits. To address these, molecular information about the genes and the subunits is required. This is so far available for subunits I11 (cytochrome cl), IV (cytochrome b) and VI-VIII [6-91 (Maarse, unpublished results). We describe here an analysis of the nuclear gene for subunit 11. Comparison of the nucleotide sequence with a partial amino acid sequence of the N terminus of the mature protein reveals an amino-terminal extension which displays several features common to the presequences of other imported mitochondrial proteins. The remaining protein sequence is predominantly hydrophilic, consistent with previous suggestions that the bulk of the subunit protrudes from the mitochondrial inner membrane into the matrix space MATERIALS AND METHODS Strains and mediaSaccharomyces cerevisiae strains DL1 (a, his3, leu2, ura3) and HR2 (a, his3,leu2, trpl) were used for most experiments. Transformation was carried out as described by Klebe et al. [Ill and transformants were selected on minima...

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Section: Gene Disruptionmentioning

confidence: 94%

Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

Oudshoorn

Steeg

Swinkels

et al. 1987

European Journal of Biochemistry

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“…The nuclear gene encoding the 14-kDa subunit VII of ubiquinol : cytochrome c oxidoreductase is present as a single copy in the haploid yeast genome [38]. The gene, as outlined in Fig.…”

Section: Gene Inactivationmentioning

confidence: 99%

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

Schoppink

Berden

Grivell

1989

European Journal of Biochemistry

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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.

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“…The chromosomal location of the SOD) gene was determined by the method of Falco and Botstein (26) as described (27 (29), blotting of DNA to nitrocellulose filters (30), preparation of labeled hybridization probes with fragments cloned into M13 mp7 (31), isolation of total cellular proteins (32), determination of protein concentrations (BCA procedure of Pierce with bovine serum albumin as the standard), protein blotting (33), and assays of lipid peroxidation as measured by monitoring malondialdehyde and hydroperoxide production (34). (Fig.…”

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confidence: 99%

A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.

Loon¹,

Pesold-Hurt²,

Schatz³

1986

Proc. Natl. Acad. Sci. U.S.A.

242

123

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The nuclear gene for manganese-containing superoxide dismutase (MnSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of yeast mitochondria was mapped on chromosome VIII and inactivated by gene disruption. The resulting mutant lacked any protein cross-reacting with antiMnSOD antibodies, and its mitochondria exhibited less than 1% of the cyanide-insensitive superoxide dismutase activity found in mitochondria of the wild-type parent strain. In the absence of oxygen, the mutant grew as rapidly as the wild-type parent. However, increasing concentrations of oxygen led to a progressive inhibition of growth. Experiments implicated superoxide in reactions such as DNA breakage, hyaluronate depolymerization, linoleate oxidation, erythrocyte lysis, and bacterial killing (6). Prolonged exposure of animal, plant, or bacterial cells to increased oxygen concentrations causes an increase in their superoxide dismutase activity, and the increased activity correlates with an increased ability to tolerate the higher oxygen concentrations (2, 7-10). In addition, exposure of bacteria to redox dyes that promote the formation of superoxide radicals causes a dramatic induction of MnSOD (11,12).Whether superoxide dismutases indeed contribute to the cell's protection against superoxide radicals, is, however, still under debate (for reviews, see refs. 1, 2, and 13). For example, it has been argued that the superoxide-decomposing activity of superoxide dismutases is artifactual and that the true function of these enzymes might well involve metal storage or some other, as yet unknown, reaction (1 MATERIALS AND METHODSStrains and Culture Conditions. The Saccharomyces cerevisiae strains DL1 (a, leu2-3, 2-112, his3-11, 3-15, ura3-251, 3-372, 3-328) and CHL2881-1C (a, ura3, leu2, trpl, Abbreviations: MnSOD, manganese-containing superoxide dismutase; kb, kilobase(s). 3820The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes

Cited by 9 publications

References 20 publications

Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

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

A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.

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Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes

Cited by 9 publications

References 20 publications

Subunit II of yeast QH2:cytochrome‐c oxidoreductase

Subunit II of yeast QH2:cytochrome‐c oxidoreductase

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

A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.

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Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

Subunit II of yeast QH₂:cytochrome‐c oxidoreductase