Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria.
The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc1 complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212-16218]. The deletion mutants lacking amino acid residues 55-66 or residues 161-180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161-180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc1 complex; however, the protein lacking residues 55-66 was assembled in vitro into the bc1 complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55-66. This deletion mutant protein was also assembled into the bc1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55-66, is not required for assembly of the iron-sulfur protein into the bc1 complex; however, this association did not lead to enzymatic activity of the bc1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.
Ramabadran
R S
RS
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506-9142, USA.
Japa
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S
Beattie
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DS
D014450:Electron Transport Complex III / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids; D017384:Sequence Deletion; D014170:Transformation, Genetic; D015003:Yeasts / Q000378:metabolism
D005656:Fungal Proteins; D007506:Iron-Sulfur Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III