RESUMEN
Translation of mitochondrial cytochrome b RNA in yeast requires the product of the nuclear gene CBS1, a 27.5 kDa soluble mitochondrial protein. In this paper we show that the CBS1 gene is located on chromosome IV immediately adjacent to COX9, the gene coding for cytochrome c oxidase subunit VIIa. CBS1 is transcribed as a very low abundant 900 b RNA. Transcription starts at a single position 101 bp upstream of the CBS1 initiation codon. At positions -39 to -27 of its leader sequence it contains a small open reading frame of 4 codons. By monitoring the beta-galactosidase activity of a CBS1/lacZ fusion construct we show that expression of CBS1 is subjected to regulation by oxygen and by glucose: the beta-galactosidase activity is elevated threefold in glycerol or galactose grown cells compared to that in glucose grown cells. A further threefold reduction of the activity is observed in anaerobically grown cells. In accordance with this result is the observation that the steady-state level of CBS1 mRNA of anaerobically grown cells is ninefold lower than that of aerobically cultured cells.
Asunto(s)
Grupo Citocromo b/genética , Regulación de la Expresión Génica , Genes Fúngicos , Biosíntesis de Proteínas , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Anaerobiosis , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Grupo Citocromo b/biosíntesis , Complejo IV de Transporte de Electrones/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.
Asunto(s)
Grupo Citocromo b/biosíntesis , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Mitocondrias/metabolismo , Proteínas Mitocondriales , Precursores de Proteínas/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transactivadores , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Grupo Citocromo b/genética , ADN de Hongos/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Biosíntesis de Proteínas , Precursores de Proteínas/genética , ARN de Hongos/biosíntesis , Saccharomyces cerevisiae/genética , SolubilidadRESUMEN
The 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.
