Gerhard Rödel

SCO1, a yeast nuclear gene essential for accumulation of mitochondrial cytochrome c oxidase subunit II

SummaryWe have identified and isolated a novel yeast nuclear gene (SCO1) which is essential for accumulation of the mitochondrially synthesized subunit II of cytochrome c oxidase (CoxII). Analysis of the mitochondrial translation products in a sco1-1 mutant reveals a strong reduction in CoxII. Examination of mitochondrial transcripts by Northern blot hybridization shows that transcription and transcript maturation of OXI1, the gene coding for CoxII, is not affected. Therefore the SCO1 gene product must be involved in a post-transcriptional step in the synthesis of CoxII. We have isolated a 1.7 kb DNA fragment from a yeast gene bank which carries the functional SCO1 gene. Two RNA species of 0.9 kb and 1.2 kb, respectively, hybridize with this DNA fragment, which is localized on chromosome II. Cells whose chromosomal 1.7 kb fragment has been replaced by the yeast URA3 gene fail to accumulate CoxII and in addition subunit I of cytochrome c oxidase (CoxI). The possibility that the SCO1 gene product is bifunctional, i.e. required for both CoxI and CoxII accumulation, is discussed.

Two yeast nuclear genes, CBS1 and CBS2, are required for translation of mitochondrial transcripts bearing the 5'-untranslated COB leader.

SummaryMutations in one of the yeast nuclear genes CBS1 or CBS2 both prevent the excision of the maturasecoding introns bI2, bI3 and bI4 from the mitochondria) COB precursor transcript. Mutant strain MK2 (cbsl-1) has recently been reported to be primarily defective in the translation of COB transcripts, as it can be suppressed by a fusion of the COB structural gene with the 5′ untranslated leader of the mitochondrial OLI1 gene (G. Rödel, A. Körte and F. Kaudewitz, Curr Genet 9: 641–648). Here I report that the effect of mutation cbs2-1, too, is suppressed by this gene rearrangement. CBS2 is the second nuclear gene identified which is involved in the translation of mitochondrial transcripts bearing the 5′ untranslated COB leader. Gene specific translation control appears to be a major mode of regulation of mitochondria) gene expression in Saccharomyces cerevisiae.

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

SummaryThe SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a β-Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho0 strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.

Accumulation of the cytochrome c oxidase subunits I and II in yeast requires a mitochondrial membrane-associated protein, encoded by the nuclear SCO1 gene.

SummaryThe yeast nuclear SCO1 gene is required for accumulation of the mitochondrially synthesized cytochrome c oxidase subunits I and II (COXI and COXII). We cloned and characterized the SCO1 gene. It codes for a 0.9 kb transcript. DNA sequence analysis predicts a 33 kDa protein. As shown by in vitro transcription and translation experiments in combination with import studies on isolated mitochodria, this protein is matured into a 30 kDa polypeptide which is tightly associated with a mitochondrial membrane. The possible function of the SCO1 gene product in the assembly of cytochrome c oxidase is discussed.

Association of cytochrome b translational activator proteins with the mitochondrial membrane: implications for cytochrome b expression in yeast

SummaryThe products of the nuclear genes CBS1 and CBS2 are both required for translational activation of mitochondrial apocytochrome b in yeast. We report the intramitochondrial localization of both proteins by use of specific antisera. Based on its solubilization properties the CBS1 protein is presumed to be a component of the mitochondrial membrane; the detergent concentrations needed to release CBS1 from mitochondria are almost the same as for cytochrome c1. In contrast, CBS2 behaves like a soluble protein, with some characteristics of a membrane-associated protein. A model is presented for translational activation of cytochrome b, which might also be applicable to translational regulation of other mitochondrial genes.

The yeast nuclear gene CBS1 is required for translation of mitochondrial mRNAs bearing the cob 5' untranslated leader

SummaryMitochondrial translation of the cob mRNA to yield apocytochrome b is specifically dependent on the nuclear gene CBS1, while mitochondrial translation of the oxi2 mRNA to yield cytochrome oxidase subunit III (cox III) is specifically dependent on the nuclear gene PET494. Chimeric oxi2 mRNAs bearing the 5′ leaders of other mitochondrial mRNAs, transcribed from rho- mitochondrial DNAs termed MSU494, are translated in pet494 mutants. In this study, we examined translation of coxIII from MSU494-encoded chimeric mRNAs in zygotes of defined nuclear and mitochondrial genotype. CoxIII was translated from a chimeric mRNA bearing the cob leader only when the zygotes contained a wild-type CBS1 gene. CoxIII translation from an mRNA bearing the 5′ leader of the mitochondrial gene aap1 was not dependent on CBS1 activity. We conclude that the product of the nuclear gene CBS1, or something under its control, acts in the mitochondrion on the cob mRNA 5′ leader to activate translation of downstream coding sequences.

Mitochondrial suppression of a yeast nuclear mutation which affects the translation of the mitochondrial apocytochrome b transcript.

SummaryWe describe a mitochondrial suppressor mutation, which restores respiratory competence to the nuclear pet−-mutant MK2. This mutant lacks the message of the mitochondrialcob-gene and instead accumulates a partially spliced pre-mRNA which is not translated. Complete processing and translation of thecob-RNA is restored by a rearrangement of the mitochondrial DNA, leading to a fusion of thecob-coding sequences with the leader ofoli1, the mitochondrial gene coding for subunit IX of the ATPase. We conclude that the nuclear gene affected in MK2 is essential to allow translation of transcripts which contain thecob-leader sequence.

Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae.

SummaryThe respiratory deficient yeast nuclear mutant MK3 is defective in the synthesis of the mature transcripts of the mitochondrial COB and OX13 genes, which code for apocytochrome b and subunit I of cytochrome c oxidase, resp. Introns 3 and 4 of the COB transcript (bI3 and bI4) and intron 4 (aI4) of the OXI3 transcript can not be excised (Pillar et al. 1983a, b). When combined with mitochondrial genomes lacking introns bI1, bI2 and bI3, or lacking intron bI3 alone the mutant is respiratory competent. Thus, the non-excision of bI4 and aI4 turns out to be an indirect effect of the mutation. From a wild type yeast genebank a plasmid has been isolated with a 3.3 kb DNA insert, which complements the mutant. Subcloning experiments assigned the functional gene to a 1.6 kb HaeIII-Sau3A fragment. Hybridization experiments showed, that it is (i) a single copy gene, (ii) also present in strain D273-10B, containing the “short form” mitochondrial genome (lacking the COB introns bI1-bI3), and (iii) located on chromosome IX. The nuclear gene defective in mutant MK3, was named MRS1 (Mitochondrial RNA Splicing). The involvement of this nuclear gene in the excision of a single group I mitochondrial intron (bI3) of the COB transcript is discussed.

Molecular cloning of the yeast nuclear genes CBS1 and CBS2

SummaryThe yeast nuclear genes CBS1 and CBS2 are both required for translation of the mitochondrial COB transcripts. Here we report on the identification of two unique chromosomal DNA-sequences of 2 kb and 2.3 kb from yeast wild type gene banks which functionally complement cbs1 and cbs2 mutants, respectively. Disruption of the homologous DNA-fragments by insertion of the URA3 gene generates respiratory deficient cells which fail to complement the original mutants. Cells with these gene disruptions are phenotypically identical to the original cbs1 and cbs2 mutants with respect to cytochrome spectra and mitochondrial translation products. The results exclude the possibility that suppressor genes have been cloned and confirm the conclusion that both genes, CBS1 and CBS2, specifically are involved in translation of mitochondrial COB RNA.

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

SummaryThe nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5′ untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.