Respiratory chain-linked NADH dehydrogenase. Mechanisms of assembly.
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.
Department of Biochemistry, School of Medicine, Oregon Health Sciences University, Portland 97201-3098.
R E
RE
Hall
J F
JF
Hare
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D003579:Cytochrome Reductases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010101:Oxygen Consumption; D011499:Protein Processing, Post-Translational; D051381:Rats
D046911:Macromolecular Substances; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase