Figure 1

Cells that overexpress HAP4 contain more respiratory chain complexes than wild-type cells. Two-dimensional blue native gel electrophoresis of mitochondrial proteins isolated from glucose-grown wild-type (WT YPD, rich glucose medium) or HAP4-overexpressing cells (HAP4 YPD). Mitochondrial proteins from ethanol-grown, respiring wild-type cells are shown as control (WT YPEG, rich medium containing non-fermentable carbon sources: 2% ethanol and 2% glycerol). Respiratory-chain complexes V, III, IV and II from mitochondria isolated from 40 mg (wet weight) wild-type or HAP4 cells were separated in the first dimension (from left to right) by blue native, 5-13% gradient gel electrophoresis [18]. These complexes were subsequently fractionated in the second dimension (from top to bottom) by denaturing gel electrophoresis (10% SDS) and proteins in the gel were visualized by Coomassie staining. The arrow indicates a spot that was determined to be Pma1p, a non-mitochondrial, H⁺-transporting ATPase which is localized in the plasma membrane [27], and which was used as control for the amount of cells fractionated.