ASSEMBLY OF CYTOCHROME-C-OXIDASE IN CULTURED HUMAN-CELLS

The assembly of cytochrome-c oxidase was studied in human cells cultur ed in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensiona l PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subun its of cytochrome-c oxidase. Proteins were also visualized using metab olic labeling followed by two-dimensional electrophoresis and fluorogr aphy. These techniques allowed identification of two assembly intermed iates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochro me-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and eit her VIIa or VIIb.