Showing papers on "Mitochondrial biogenesis published in 1974"

Mitochondrial protein synthesis in HeLa cells.

Abstract: HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a3 and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.

Oligomycin sensitivity of ATPase studied as a function of mitochondrial biogenesis, using mitochondrially determined oligomycin-resistant mutants of Saccharomyces cerevisiae.

Abstract: The quantitative comparison of oligomycin sensitivity of ATPase from two mitochondrially determined oligomycin-resistant mutants (locus OI and OII) and the wild-type strain (all three isonuclear), as a function of mitochondrial biogenesis, gave the following results: 1 The mutations at the loci OI and OII confer distinctly different oligomycin resistance on mitochondrial ATPase, OI being more resistant than OII, and both more resistant than the ATPase from the wild-type strain. 2 In spite of these differences in oligomycin resistance, the binding of the ATPase to the mitochondrial membranes does not appear to be modified. 3 The phenotypic oligomycin resistance of mitochondrial ATPase, produced by growth of the wild type strain in the presence of chloramphenicol, is notably higher than the highest genotypic oligomycin resistance. 4 The oligomycin sensitivity or resistance of ATPase is strictly identical in anaerobic promitochondria and aerobic mitochondria, i.e. the difference between the oligomycin sensitivity of the ATPase from these strains is maintained in such extremely different physiological states. 5 In contrast, the ATPase isolated during respiratory adaptation of the wild-type strain had distinctly higher oligomycin resistance than the promitochondria or the aerobic mitochondria. This “transient” resistance displayed by the wild-type ATPase during adaptation (in the absence of growth) was very similar to the genotypic resistance of the ATPase from the mutant at locus OI.

Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver.

Abstract: The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

Symposium on Non-chromosomal Inheritance: XIII International Congress of Genetics THE USE OF HYBRID SOMATIC CELLS AS AN APPROACH TO MITOCHONDRIAL GENETICS IN ANIMALS

Abstract: We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time. The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are moist likely covalent bonds. This linkage may be described as “recombination” of mtDNA sequences from two different animals ITOCHONDRIA from all organisms studied contain DNA; this DNA is required for the formation of functional mitochrondria (see BORST 1972). Therefore, we may regard the mitochondrial DNA (mtDNA) as the carrier of a mitochondrial genome. The study of the function, regulation, and transmission of this genome is currently an active field. The experience with bacteria and bacteriophages clearly showed that studies of this type are greatly aided by the availability of genetic tools which, when combined with biochemical analysis, will lead to the elucidation of the mechanisms involved. Genetic tools are available for the study of mitochondrial biogenesis in yeast (DUJON, SLONIMSKI and WEILL 1973; SAGER 1972; WILKIE 1972; BOLOTIN et al. 1971), and to a lesser extent in Neurospora (see SAGER 1972) and Paramecium (BEISSON and BEALE 1973). In multicellular animals the choice of genetics tools for this purpose is extremely limited. Attempts are being made at present to improve this situation in several ways: One aspect of these studies is the search in cultured cells for mutants (or variants) for mitochondrial functions. The most interesting mutants would be those which showed a cytoplasmic mode of inheritance. The search €or cell strains resistant to certain antibiotics is a promising approach since cytoplasmically-inherited mutants resistant to chloramphenicol and similar drugs have been found in yeast (THOMAS and WILKIE 1968; LINAbbreviations mtDNA mitochondnal DNA, mt-rRNA mtochondnal nbosomal RNA, =RNA complementary RNA, referring to RNA transcnbed In vitro mth Eschenchza col, RNA polymerase, mt cRNA cRNA transcribed from mtDNA as template, nDNA nuclear DNA Present address Institute of Virology, Universlty of Wurzburg, Wurzburg, Germany.