Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria

• Published in: Journal of bioenergetics and biomembranes 1997-02, Vol.29 (1), p.45-54
• Main Authors: Ramabadran, R S, Japa, S, Beattie, D S
• Format: Article
• Language: English
• Subjects: Amino acids; Cytochrome; Electron Transport Complex III - metabolism; Enzymatic activity; Fungal Proteins - metabolism; Iron sulfides; Iron-sulfur proteins; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Mitochondria; Mitochondria - metabolism; Plasmids; Residues; Sequence Deletion; Sulfur; Transformation, Genetic; Yeasts; Yeasts - metabolism
• ISSN: 0145-479X; 1573-6881
• DOI: 10.1023/A:1022459722020

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.