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The fidelity of mitochondrial translation has been examined in isolated yeast mitochondria incubated in an optimized protein-synthesizing medium (McKee, E. E., and Poyton, R. O., (1984) J. Biol. Chem. 259, 9320-9331). These studies have revealed: that isolated mitochondria synthesize bona fide mitochondrial gene products which are identical in kind and relative amounts to those synthesized in vivo; that mitochondria isolated from both mitochondrial mit- mutants and nuclear Pet mutants, which retain the capacity for mitochondrial protein synthesis, produce a mutant pattern of mitochondrial gene products which is similar to that produced in vivo; and that isolated mitochondria synthesize up to 7% of their protein mass in vitro at a rate of about one polypeptide bond/polypeptide chain/s. These studies also reveal that isolated wild type yeast mitochondria are competent in all steps in mitochondrial translation, including initiation. Using pactamycin as a specific inhibitor of translational initiation we have demonstrated that polypeptide chain initiation continues throughout a 60-min incubation period. By using this in vitro system to calculate the stoichiometry of synthesis of the major proteins coded by yeast mitochondrial DNA we have found that the var1 polypeptide is synthesized at a level which is significantly lower than all other mitochondrial gene products and that cytochrome c oxidase subunits I, II, and III and ATPase subunit 9 are synthesized in nearly equimolar amounts. These results suggest that the synthesis of these four gene products is controlled coordinately.

An in vitro mitochondrial protein-synthesizing system, which makes use of intact yeast mitochondria, has been developed in order to study mitochondrial gene expression and its control by nuclear-coded proteins. Studies with this system have revealed that: isolated mitochondria synthesize polypeptide gene products which can be radiolabeled to high specific radioactivities when incubated in a "protein-synthesizing medium" that has been optimized with respect to each of its components; two energy-generating systems, endogenous oxidative phosphorylation and an exogenous ATP-regenerating system, support the highest level of protein synthesis; and the omission of an oxidizable substrate results in the synthesis of two new polypeptides (19.5 and 18 kDa) and a decrease in the amounts of cytochrome c oxidase subunits I and II which are synthesized. They have also revealed that added adenine and guanine nucleotides increase the overall level of protein synthesis and that the added guanine nucleotides facilitate polypeptide chain elongation. Although isolated mitochondria which have been optimized for protein synthesis synthesize normal gene products (McKee, E. E., McEwen, J. E., and Poyton, R. O., (1984) J. Biol. Chem. 259, 9332-9338) they still respond to an added dialyzed S-100 fraction from yeast cells by increasing their level of protein synthesis. This stimulation is observed in the presence of optimal concentrations of GTP, making it unlikely that guanyl nucleotides or enzymes which synthesize them are the sole stimulatory factors present in cellular cytosolic fractions, as suggested by Ohashi and Schatz (Ohashi, A., and Schatz, G. (1980) J. Biol. Chem. 255, 7740-7745).