Analysis of Proteins Synthesized in Mitochondria of Cultured Mammalian Cells. An Assessment of Current Approaches and Problems in Interpretation

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Abstract
The conditions which enable highly efficient utilization of [35S]methionine by cultured mammalian cells [HeLa B human cervical cancer cells, D98/AH-2 human sternal marrow cells, BREL human cells, 1R mouse cells and RAG rat adenocarcinoma cells] and the resolution of selectively labeled mitochondrial products are described. Analysis of mitochondria purified from cells labeled in the presence or absence of inhibitors of cytoplasmic (or mitochondrial) protein synthesis indicated that about 5% of the [35S]methionine incorporated into mitochondrial proteins results from synthesis on mitoribosomes. The electrophoretic profile of the detergent-solubilized proteins of mitochondria isolated from cells which were labeled in the presence of 50 .mu.g/ml emetine was similar to those obtained with extracts prepared by direct solubilization of the intact cells after incorporation of label. Pulse-labeling 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. Erythromycin suppressed the snythesis of additional labeled products which were detected in extracts of 1 cell line, when analyzed by procedures which normally detected only mitochondrially synthesized proteins. These additional bands were attributed to the synthetic activity of Mycoplasma.