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

Abstract

The conditions which enable highly efficient utilization of [35S]methionine by cultured mammalian cells and the resolution of selectively labelled mitochondrial products are described. 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 [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 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. 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. 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.

Synthesis of mitochondrial proteins in isolated rat hepatocytes

“…Electrophoretic analysis of the mitochondrially-translated peptides indicated that these products were stable after 3 h chase, and that the relative distribution of radioactivity between the various peaks did not differ significantly from that shown in fig.3. These findings again provide no support for an interconversion between the peptides [7]. They also indicate that breakdown of newly synthesized proteins is negligible under the conditions of our experiments.…”

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

“…Whole cell lysates (40 μ g) were electrophoresed through 12% SDS-PAGE. The gels were dried on 3MM chromatography paper (Whatman), and the total intensities of the signals were quantified by phosphorimaging analyses (Jeffreys and Craig, 1976). The siRNA mediated knock-down efficiency of the corresponding mitochondrial ribosomal protein was confirmed with immunoblotting analysis of whole cell lysates or crude ribosomal fractions, prepared as stated above.…”

Long-Term Consequences of Radiation-Induced Bystander Effects Depend on Radiation Quality and Dose and Correlate with Oxidative Stress

“…Mitochondrial protein translation was assessed as described (29) by incorporation of 35 S-methionine in cells wherein cytoplasmic protein synthesis was inhibited by cycloheximide. Briefly, confluent cell cultures were washed twice in PBS, incubated in cysteine- and methionine-free Dulbecco’s Modified Eagle Medium (Cellgro) for 10 min at 37°C under an atmosphere of 5% CO 2 in air and subsequently incubated for 1 h with cycloheximide (10 μg/ml).…”