Formation of the Yeast Mitochondrial Membrane

Amino acid incorporation in vitro by isolated yeast mitochondria could be supported by either an external ATP‐regenerating system or by the respiratory‐chain‐linked synthesis of ATP. However, the incorporation rates obtained with ATP and succinate were almost double those obtained with the ATP‐regenerating system and were inhibited by either antimycin A or oligomycin. In either system, addition of chloramphenicol, erythromycin or ethidium bromide caused an 80–90% inhibition of incorporation, actinomycin D and cordycepin caused a 20% inhibition, while rifamycin or cycloheximide had no effect. The rate of amino acid incorporation in vitro by isolated mitochondria was not subject to glucose derepression. In contrast, the rate of cycloheximide‐insensitive protein synthesis into mitochondrial membranes in vivo was lowered over 90% in repressed cells. Analysis of the labeled mitochondrial membranes by sodium dodecylsulfate gel electrophoresis indicated that the products of mitochondrial protein synthesis were qualitatively identical, but quantitatively different, in glucose‐repressed and derepressed cells. For example, a two‐fold increase in labeling into both the two peaks of highest molecular weight as well as the peak of lowest molecular weight was observed in mitochondrial membranes obtained from repressed cells as compared to mitochondrial membranes obtained from partially and fully derepressed cells. The rate of amino acid incorporation in vitro as well as the labeling of mitochondrial membranes in vivo was stimulated 50—100% in partially derepressed yeast cells which had been grown in medium containing chloramphenicol for three hours, washed, and allowed to grow for one hour in fresh medium without chloramphenicol. The stimulation of labeling in vivo resulted in a threefold increase of labeling into the peak of lowest molecular weight with a concomitant decrease of labeling into the peaks of high molecular weight suggesting that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins. Furthermore, growth of the cells in the presence of cycloheximide, which would result in a depletion of the pool of mitochondrial proteins synthesized in the cytoplasm, followed by growth in fresh medium without cycloheximide, resulted in a 50% decrease in the incorporation rate in vitro.