Biogenesis of mitochondria. 52

Summary The characteristics of recombination of several petite (rho ^-) mutants of S. cerevisiae that retain the -influenced region of the mitochondrial genome, identified by the markers cap1-r, ery1-r and tsr1, are described. The petites were derived from an ^– grande (rho ⁺) strain and those petites which retain all three markers show recombination properties similar to those of the ^- parental strain. However, other rho ^- mutants that retain the cap1 and ery1 loci but have lost the tsr1 locus, which is located between cap1 and ery1, show markedly different properties of mitochondrial transmission and recombination, consistent with the presence of ⁺ alleles. The association of an internal deletion between the cap1 and ery1 loci with a change in phenotype provides additional evidence for the location of between these two loci.Although the petites deleted for the tsr1 locus exhibited the recombination properties of ⁺ strains, it was not possible to transmit this characteristic to rho ⁺ recombinant cells. Experiments on the kinetics of elimination by ethidium bromide of the cap1 and eryl markers from the petites and measurements of the buoyant densities of their mtDNA species did not indicate major changes (such as selective sequence repetition) in the sequences of the mtDNAs. The possible nature of the changes in the mtDNAs of these petites is discussed in light of recent studies on the physical nature of the alleles.     

Protein sorting in mitochondria.

Most polypeptides that are imported into the mitochondrial matrix use a common translocation machinery. By contrast, proteins of the other mitochondrial compartments are imported by a variety of different mechanisms. Some of these proteins completely bypass the common translocation machinery, others use only the outer membrane components of this machinery, and still others use components of this machinery from both the outer and inner membranes. Import to the intermembrane space compartment provides examples of all three possibilities.     

Biogenesis of mitochondria. 53

Summary As shown by gel electrophoresis analysis, E. coli mutant 219 is mutated on the gene coding for S4. This mutant and the parental strain have been studied at the permissive (30°) and the non-permissive temperature (42°) for ribosome assembly and r-protein biosynthesis.The extracts of cells grown at the non-permissive temperature were analyzed by sucrose gradients: Particles sedimenting more slowly (28S) than normal 30S accumulate while 50S precursors undergo maturation and attach to the preformed 30S subunits yielding 70S ribosomes. In addition a small but detectable amount of 30S is also synthesized at 42°. The 28S particles contain all 30S r-proteins except S1, S2 and S12; S5, S7 and S21 are present in reduced amount.The relative rate of biosynthesis of individual r-proteins was determined by pulse-labelling the cells with radioactive leucine. Individual r-proteins were purified from cell extract by the three-dimensional gel electrophoresis technique. The relative rate of biosynthesis of 50S proteins is unchanged in mutant cells grown at 42°. Only the rate of synthesis of five 30S proteins is modified by the temperature shift: S10, S13, S20 and S21 have an increased rate, while S18 is synthesized at a reduced rate. Thus in cells deficient in the assembly of 30S subunits, although the biosynthesis of a few 30S r-proteins is specifically altered, the synthesis of most r-proteins appears to be controlled in the same way as are total cell proteins.     

Biogenesis of mitochondria. 30

Summary Mitchondrial gene recombination in S. cerevisiae was investigated using four combinations of mitochondrial markers: [oli1-r ery1-r], [oli1-r spi2-r], [oli1-r spi3-r] and [oli1-r spi4-r] in cis bifactorial crosses to [oli-s ery-s spi-s] strains. A number of sensitive strains including representatives of both mating types and of diverse origin were used. The crosses were analysed for frequency and polarity of mitochondrial gene recombination as well as the frequency of transmission into the diploid progeny of individual mitochondrial determinants.The results show that the polarity of recombination varied markedly in crosses between a single pair of mitochondrial markers and many unrelated sensitive strains. For example, one series of crosses included polarity values of 1.7,0.34,0.081, and 0.021. Furthermore, there was also considerable variability in frequency of recombination and frequency of transmission of individual markers and these frequencies were not correlated in many cases with polarity values. However, in certain other crosses involving different marker combinations there was a correlation between extreme polarity, high recombination frequency and high transmission frequency of one marker. The results are not compatible with polarity being determined by a simple mitochondrial sex factor and suggest that several different interactions are operating which might include nuclear phenomena.     

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.     

Light-dependent ATP synthesis in mitochondria.

Light-dependent ATP synthesis was studied in an illuminated suspension of rat liver mitochondria. The action of light was shown to lead to an increase in the ATP content in the absence of oxidisable substrates and in the presence of high (hundreds of microM) ADP concentrations in the medium. At a relatively low (50 microM) ADP concentration, efficient light-dependent phosphorylation was observed in the presence of alpha-ketoglutarate. Prolonged illumination stimulated ATP hydrolysis. Rotenone, antimycin, azide, dicyclohexylcarbodiimide, and oligomycin inhibited the light-dependent phosphorylation almost completely. The level of ATP decreased under the action of 2,4-dinitrophenol in the dark but was restored by high light intensities. Blue light, 436 nm, was most efficient to produce light-dependent phosphorylation. It is assumed that quanta of vibrational excitation formed in the course of vibrational relaxation and the internal conversion of photoexcited flavoproteins and cytochromes are transferred to the ATP-synthetase and "eject" ATP from the active center, thus shifting the enzymatic reaction to ATP production.     

Interaction theory of mammalian mitochondria.

We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit.     

Pyruvate transport by thermogenic-tissue mitochondria.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.