Proteins Coded by Mitochondrial DNA of Mammalian Cells

Abstract

Improved conditions are described for the maintenance of protein synthesis by isolated liver mitochondria. The products of synthesis in vitro are first attached to a 55-S mitoribosome (mitochondrial-ribosome) but move off the mitoribosome as completed peptide chains in less than 15 min a t 30 "C. Over a 1-h period of incubation there is no change in the spectrum of molecular weights of the peptides being synthesized. The protein synthesized by isolated hamsterliver mitochondria were compared with those synthesized by whole cells under conditions where cytoplasmic protein synthesis had been blocked by emetine. The proteins synthesized by whole cell mitochondria were indistinguishable from those synthesized by isolated mitochondria. By the combined use of emetine, chloramphenicol and ethidium bromide as specific inhibitors of cytoribosomal and mitoribosomal protein synthesis it was possible to calculate that 7 of the total mitochondrial protein was coded by the mitochondrial genome. A phosphate buffer a t p H 11.5 solubilized about goo/, of the total protein of the mitochondrion but left over goo/, of the protein coded by the mitochondrial genome as an insoluble residue. This fraction on polyacrylamide in the presence of sodium dodecylsulphate and mercaptoethanol was shown to contain a t least 20 distinct proteins. At least 10 of these were coded by the mitochondrial genome and their molecular weights ranged from 14000 to 50000. These proteins together with the mitochondrial tRNA and the mitoribosomal RNA account for most of the information available in the mitochondrial genome.Investigations in several laboratories (see review, Ashwell and Work [l]) have shown that mitochondria are semi-autonomous bodies which contain informational DNA, mitoribosomes (mitochondrial ribosomes) and a mechanism for protein synthesis which is similar to but quite distinct from that present in cytoplasm of the same cells.It has been shown that the 16-S and 12-S RNA of mitoribosomes is coded by mitochondrial DNA, as are an uncertain number of transfer RNA species[a]. The molecular weights of various types of mammalian mitochondrial DNA molecules are all about 10000000, and the DNA is a double-stranded circular molecule, only one strand of which seems to be informational [3]. Hybridization experiments have indicated that the genes for mitoribosomal RNA are not reiterated on the mitoribosomal DNA [3] so that some of the mitochondrial genes are certainly available to code for messenger RNA. This messenger RNA appears to code for proteins which form a n essential part of the structure of the inner mitochondrial membrane and in their absence the enzymes of electron transport, which are coded by nuclear genes, accumulate in the cytoplasm and cannot be integrated into their active configuration in the cristae membrane. It thus appears that the mitochondrial Enzyme. Lactate dehydrogenase (EC 1.1.1.27); pyruvate kinase (CE 2.7.1.40); lyoszyme (EC 3.2.1.17).genes code for organizer proteins essential to the life of the cell [l].It has been our p...

Stable and Labile Products of Mitochondrial Protein Synthesis in vitro

“…Coote and Work [46] concluded to identical patterns of synthesis in vitro and in vivo in BHK-1 cells, both products being in the molecular weight range 23000-43000. Similar findings for rat liver are reported by Ibrahim et al [46]. Tzagoloff and Akai [47] studying the product in vivo in yeast found that its electrophoretic behaviour in dodecylsulfate polyacrylamide gels depended entirely on the method of preparation of the product.…”

Mitochondrial Protein Synthesis in a Mammalian Cell-Line with a Temperature-Sensitive Leucyl-tRNA Synthetase

“…They are also similar to the profiles of proteins labelled in isolated mitochondria from HeLa cells [31], BHK-21 cells [32] or rat liver [33]. In general, the proteins are often found as two or three poorly resolved classes having apparent molecular weights of less than 50000.…”

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

“…Somewhat lower rates of mitochondrial protein synthesis in the presence of various cytoplasmic inhibitors have been found by Perlman and Penman [20] and Brega and Baglioni [21] for HeLa cells incorporating labelled leucine. Any direct inhibitory effect of emetine or cycloheximide on mitochondrial protein synthesis [18,22] [23]) is probably too low since (a) the mitochondria analysed were not completely pure (see Table 1 and Fig. 6), and (b) cycloheximide might have some direct and/or indirect inhibitory effect on mitochondrial protein synthesis.…”