Formation of the Yeast Mitochondrial Membrane

The kinetics of formation of different enzyme complexes of the inner mitochondrial membrane were studied in yeast undergoing derepression during growth in 5% glucose. Each different enzymic activity started to increase at a unique time in the growth curve, increased at a characteristic rate and reached a maximum at a different time. For example, oligomycin‐sensitive ATPase activity increased from the earliest time studied (7 h) reaching a maximum at 17 h which corresponds to early stationary phase. Succinate dehydrogenase and succinate‐cytochrome c reductase activities increased throughout the 24‐h growth period with no appreciable lag. In contrast, a lag of 13 h occurred before NADH dehydrogenase and NADH‐cytochrome c reductase activities started to increase. NADH‐dehydrogenase activity reached a maximum at 17 h, while the activity of NADH‐cytochrome c reductase increased throughout the growth period. The last enzymic activity to be derepressed was cytochrome oxidase which did not start to increase until about 14 h of growth and then continued to increase throughout the growth period. These results suggest that the various enzyme complexes of the mitochondrial membrane are assembled into the membrane in an asynchronous process. The inhibitors of protein synthesis, cycloheximide and chloramphenicol, were added at different times during the growth cycle. The appearance of succinate and NADH dehydrogenases, as well as succinate‐cytochrome c reductase was completely blocked by the addition of cycloheximide at all times but unaffected by chloramphenicol suggesting that these enzymes do not contain products of mitochondrial protein synthesis. The increase in ATPase activity was completely inhibited by the addition of both inhibitors at all times indicating that formation of a functional ATPase complex requires the concomitant synthesis of proteins on both sites. Addition of chloramphenicol to repressed cells blocked the appearance of both cytochrome oxidase and NADH‐cytochrome c reductase but activity of these enzymes continued to increase for several hours after addition of cycloheximide at this time. These results suggest that synthesis of mitochondrial proteins on cytoplasmic ribosomes may precede that on mitochondrial ribosomes. When either inhibitor was added to partially derepressed cells, activity of both cytochrome oxidase and NADH‐cytochrome c reductase increased for several hours and then ceased suggesting that mitochondrial proteins synthesized both in the cytoplasm and in the mitochondria accumulate to a limited extent prior to integration into a functional unit in the membrane.