Analysis of Proteins Synthesized in Mitochondria of Cultured Mammalian Cells

1 The conditions which enable highly efficient utilization of [ 35 S]methionine by cultured mammalian cells and the resolution of selectively labelled mitochondrial products are described. 2 Analysis of mitochondria purified from cells labelled in the presence or absence of inhibitors of cytoplasmic (or mitochondrial) protein synthesis indicated that about 5% of the [ 35 S]Methionine incorporated into mitochondrial proteins results from synthesis on mitoribosomes. 3 The electrophoretic profile of the detergent‐solubilized proteins of mitochondria isolated from cells which were labelled in the presence of 50 μg/ml emetine was similar to those obtained with extracts prepared by direct solubilization of the intact cells after incorporation of label. 4 Pulse‐labelling studies suggested that the components resolved by electrophoresis and autoradiography under the conditions described, apparently represent discrete and stable end products of mitochondrial protein synthesis. No post‐synthetic modification or degradation of these products was detected. 5 Erythromycin was found to suppress the synthesis of additional labelled products which were detected in extracts of one cell line, when analysed by procedures which normally detected only mitochondrially synthesized proteins. These additional bands were attributed to the synthetic activity of Mycoplasma.