Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes

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Abstract
The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labeled with L-[35S]methionine. Labeled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labeled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labeling. Subunit I was heavily labeled, however, in intact mitochondria after 10 min. A similar pattern of labeling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labeled methionine. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labeling pattern described above. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.