Studies on the Biogenesis of Mitochondrial Protein Components in Rat Liver Slices

Mitochondria were isolated from rat liver slices, which had been incubated with (14C)leucine, and fractionated into water-soluble, KCl-soluble, and KCl-insoluble proteins. After a 30-min incubation, all fractions contained radioactivity. Addition of an excess of unlabeled leucine at this time followed by another 30- to 90-min incubation caused a cessation of incorporation into the KCl-insoluble fraction; however, the specific activity of the soluble fractions increased 40 to 90% during this time. Labeling of the water-soluble proteins reached its maximum 30 min after addition of the chase, while the KCl-soluble proteins continued to increase at a linear rate for 90 min after chase addition. The addition of puromycin after a pulse label gave identical results. In time studies, (14C)lysine was incorporated into the KCl-insoluble proteins after 15 min. A lag of 30 min occurred before significant incorporation was observed in the soluble proteins. Chloramphenicol (0.6 to 1.2 mm) inhibited incorporation into the KCl-insoluble proteins but had little effect on that of the soluble proteins. The inhibition of leucine incorporation into the KCl-insoluble proteins and into isolated mitochondria in vitro was specific for the d isomer of chloramphenicol. High concentrations of either the d or l isomer of chloramphenicol inhibited labeling of all of the mitochondrial fractions and the microsomes. This inhibition appeared to result from an effect on respiration since, in isolated mitochondria, respiration of DPNH-linked substrates is inhibited equally by these concentrations of the d or l isomer of chloramphenicol. During short term experiments at low temperatures, cycloheximide inhibited incorporation into the KCl-insoluble proteins less than that into the soluble proteins or microsomes. These results are consistent with the hypothesis that the biogenesis of mitochondria involves the initial synthesis of certain insoluble proteins by the mitochondria and the subsequent integration of soluble protein synthesized outside the mitochondria.