Abstract
The assembly of mitochondrially and cytoplasmically translated subunits of NADH dehydrogenase in the inner mitochondrial membrane was studied in rat hepatoma cultures. A polyclonal antibody to the purified bovine heart holoenzyme, which reacted with comigrating proteins of both rat liver and hepatoma mitochondria on immunoblots, precipitated 25-30 [35S]methionine-labeled proteins from hepatoma cell lysates. Six of these were sensitive to an inhibitor of mitochondrial translation (chloramphenicol), resistant to an inhibitor of cytosolic translation (cycloheximide), and were not present in cytochrome oxidase. By these criteria, six NADH dehydrogenase subunits are identified as being translated on mitochondrial ribosomes. The metabolic properties of the three most prominent of these at 51, 43, and 11 kDa were studied in more detail. Mitochondrial and nuclear-coded polypeptides assemble into NADH dehydrogenase at different rates as measured by incorporation of pulse-labeled proteins into immunoprecipitable enzyme. Nuclear-coded, imported polypeptides appear immediately after a pulse with [35S]methionine and retain constant stoichiometry. Mitochondrially coded proteins, although rapidly translated, appear at peak levels at different times between 0 and 12 h of chase in the immunoprecipitated enzyme. Ongoing synthesis and import of nuclear-coded proteins is necessary for mitochondrially coded proteins to be assembled. Excess, unassembled mitochondrially translated subunits are degraded in an oligomycin-sensitive manner. These data are consistent with a model in which a scaffold of imported proteins forms the inner core of the enzyme, and later arriving mitochondrially translated proteins attach to the scaffold in a time-dependent manner.
Highlights
The assembly of mitochondrially and cytoplasmically translated subunits of NADH dehydrogenase in the inner mitochondrial membrane was studied in rat hepatoma cultures
NADH DH prepared from bovine heart mitochondria and used throughout this study is nearly identical to a highly purified sample given us by Hate6 (Fig. IA, lanes 1 and 2). [3”S]Methionine-labeled polypeptides precipitated by antisera to this preparation from hepatoma mitochondrial lysates (Fig. IA, lone 3) are about the same in number and include many comigrating proteins
The similar pattern of the high molecular weight polypeptides probably reflects the evolutionary conservation of the redox groups of the enzyme, while variations in the smaller subunits between speciesis not surprising, as these are believed to play a structural role in the holoenzyme (Ragan, 1980)
Summary
DH was prepared from fresh bovine heart as described (Hatefi and Rieski, 1967). examined for contaminating cytochromes by difference spect;oscopy, and for polypeptide content by SDS-PAGE. Preparation of Mitochondria-Hepatoma mitochondria were prepared on ice from 80-95% confluent cultures on lo-cm dishes by scraping cells into 1 ml of homogenization buffer (270 mM mannitol, 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4). The supernatant was removed following centrifugation at 700 x g for 10 min, and the cell pellet washed in 0.2 ml of homogenization buffer, recentrifuged as before, and the supernatants combined. 1mrr&oprecipitation-Preimmune sera (20 11) was added to mitochondria lysed in 1% Triton X-100, 0.5% Na-deoxycholate, 2 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline (except where noted) for 1-h. The antigen-antibody complex was precipitated by incubation with 0.05 ml of Pansorbin for 1 h, and washed successively with NET containing 1% Nonidet P-40, 0.5 M NaCl, 1 mg/ml ovalbumin, and 2 mM methionine, pH 8.2; NET with 1 mg/ml ovalbumin, pH 7.2 and NET alone. Washes were repeated and immunoreactive bands were visualized using horseradish peroxidase color development reagent, containing 4-chloro-1-naphthol (Bio-Rad)
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