Identification and characterization of cytochrome bc(1) subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc(1) subunits

Abstract

We have examined the status of the cytochrome bc(1) complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc(1) complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc(1) complex was detected as a mixed population of enzymes, consisting of cytochrome bc(1) dimers, and ternary complexes of cytochrome bc(1) dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc(1) dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc(1) subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc(1) subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c(1) associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc(1) subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc(1) complex assembly.