The alpha subunit of the mitochondrial ATP synthetase is mitochondrially made in the unicellular heterotrophPrototheca zopfiii

Summary Prototheca zopfii is a unicellular, colorless alga which grows heterotrophically on several different carbon sources.P. zopfii cells were grown, labeled with [³⁵S] sulfate in the presence of cycloheximide, and the mitochondrial fraction isolated and analyzed by gel electrophoresis and autoradiography. Cycloheximide resistant translation products included a heavily labeled protein with an apparent molecular weight of 60,000. This labeled protein co-fractionated with mitochondria when the mitochondria were purified on Percoll gradients. When F₁-ATPase was isolated from the mitochondria, an active ATPase preparation was obtained which, when analyzed by gel electrophoresis, was found to contain 2 prominent proteins in the 55 kd to 60 kd region of the gel. Autoradiography of this electrophoretically-resolved ATPase preparation revealed that the larger of the two, bands, but not the smaller, was heavily labeled with [³⁵S]. Extraction of ATP synthetase (F₁ -F₀) from mitochondria with the detergent octylglucoside, yielded a preparation containing, as major components, the same two 55 kd to 60 kd proteins seen in the F₁-ATPase preparation. Limited proteolysis of the larger protein, isolated from mitochondria that had been labeled in the presence of cycloheximide, was carried out usingStaphylococcus aureus V8 protease or papain. The pattern of peptide fragments observed by Coomassie Blue staining was essentially identical to that observed by autoradiography. This shows that the labeled protein corresponds to the alpha subunit of the ATPase. Thus the alpha subunit of the mitochondrial ATP synthetase ofP. zopfii is synthesized in the mitochondria.