Biogenesis of mitochondria. Two-dimensional electrophoretic analysis of mitochondrial translation products in yeast

Abstract

1. Mitochondrial translation products of yeast Saccharomyces cerevisiae were separated according to charge as well as molecular weight by a highly resolving two dimensional electrophoretic technique (isoelectric focusing in the first dimension and SDS-electrophoresis in the second dimension). 2. The major protein components (the oligomeric form of subunit 9 of mitochondrial ATPase, var 1, cytochrome oxidase subunits I, II and III, subunit 6 of mitochondrial ATPase and cytochrome b apoprotein) were identified either from their mobility in SDS-electrophoresis or by using mit − mutants defective in certain mitochondrially made polypeptides. 3. This method allowed the separation of subunit III of cytochrome oxidase and subunit 6 of mitochondrial ATPase which cannot be resolved by conventional SDS-polyacrylamide gel electrophoresis. 4. Subunit II of cytochrome oxidase resolves in two spots of similar p I values and subunit 6 of mitochondrial ATPase resolves in two spots of similar molecular weights. In both cases the double spots disappear simultaneously following a single mutation in the corresponding structural gene. 5. Total mitochondrial proteins were also resolved two-dimensionally revealing over 100 components. The mitochondrial translation products, with the exception of subunit 9 of mitochondrial ATPase, could be easily recognized among the other mitochondrial proteins.