The role of the membrane-spanning and extra-membranous regions of the iron–sulfur protein in its assembly into the cytochrome bc1 complex of yeast mitochondria

Abstract

The assembly of six deletion mutants of the Rieske iron–sulfur protein into the cytochrome b c 1 complex was investigated by immunoprecipitation from detergent-solubilized mitochondria with specific antisera against either the iron–sulfur protein or the intact cytochrome b c 1 complex. After import, the mutant proteins lacking residues 41–55 or 66–78, located at the membrane-spanning region of the protein, and residues 182–196 located at the C-terminus of the protein, were assembled in vitro into the b c 1 complex approximately 50% as effectively as the wild type iron–sulfur protein suggesting that these regions of the iron–sulfur protein may not be critical for the assembly. By contrast, only trace amounts of the mutant proteins lacking residues 80–95, 122–135, 138–153 located in the extra-membranous region of the iron–sulfur protein were assembled into the b c 1 complex. After import in vitro into mitochondria isolated from a cytochrome b-deficient yeast strain, the mutants lacking residues 41–55 and 182–196 were assembled as efficiently as the wild type; however, the mutants lacking residues 55–66 and 66–78 were assembled less efficiently in the absence of cytochrome b suggesting that the hydrophobic membrane-spanning region, residues 55–78, of the iron–sulfur protein, may interact with cytochrome b during the assembly of the b c 1 complex.