Jordan Kolarov

ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae

All three genes (AAC1,AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator‐protein nor carrier‐mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non‐detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.

Obligatory requirement of intramitochondrial ATP for normal functionning of the eucaryotic cell

Abstract Mass formation of respiration-deficient mutants takes place in the aerobic culture of S.cerevisiae when the synthesis of ATP in mitochondria is prevented by inhibiting respiration and, simultaneously, the influx of ATP from cytosol into mitochondria is blocked by bongkrekic acid. In addition, the multiplication of the respiration-deficient mutants in complex glucose medium is arrested by bongkrekic acid. Thus, continual presence of ATP inside mitochondria is necessary for normal replication of mitochondrial DNA and also for a function related to cellular multiplication.

Anaerobic growth and formation of respiration-deficient mutants of various species of yeasts

Yeast Saccharomyces cerevisiae has often been used in the study of biogenesis and function of mitochondria taking advantage of the abilities of this species to grow under anaerobic conditions and to give rise to cytoplasmic respiration-deficient mutants. It has been usually assumed that both abilities are related to the high fermentation capacity of this yeast which can furnish energy for growth under conditions when oxidative phosphorylation is not functioning. However, this assumption has not yet been subjected to unequivocal experimental tests. Little is known of how other yeast species behave in this respect except that many species of ‘petite negative’ yeasts do not form cytoplasmic respirationdeficient mutants [ 1,2]. The use of other yeast species may help to clarify this problem and also provide new experimental possibilities for the study of mitochondria not encountered with the common baker’s or brewer’s yeasts. By employing a number of different yeast species this paper shows that the ability of a cell to grow under strictly anaerobic conditions is not related to its respiration or fermentation capacities and that these capacities are also not directly responsible for the ability of some species to form respirationdeficient mutants.

Some properties of coupled hepatoma mitochondria exhibiting uncoupler-insensitive ATPase activity

Summary Coupled mitochondria of Zajdela hepatoma exhibited ATPase activity which was not stimulated by uncouplers. In spite of this fact the properties of respiration, energy-dependent Ca2+ uptake and adenine nucleotide translocation as well as electrophoretic pattern of membrane proteins of Zajdela hepatoma mitochondria did not differ from those of Ehrlich ascites tumour mitochondria which possess uncoupler-sensitive ATPase.

Bongkrekic acid sensitivity of respiration-deficient mutants and of petite-negative species of yeasts.

Abstract 1. Growth on glucose of cytoplasmic respiration-deficient ( ρ − ) mutants isolated from five strains of Saccharomyces cerevisiae and one strain of Saccharomyces carlsbergensis were arrested by the inhibitor of mitochondrial adenine nucleotide translocation, bongkrekic acid. This indicates that the mitochondrial adenine nucleotide translocation system is preserved and necessary for growth in a number of independent ρ − mutants. 2. Growth of three “petite-negative” yeast species was arrested by a combined inhibition of respiration by antimycin A and of adenine nucleotide translocation by bongkrekic acid. Thus, the arrest of growth upon inhibition of adenine nucleotide translocation in non-respiring cells is not specific for ρ − mutants and may be a general characteristic of eucaryotic cells.

Oxidative phosphorylation in yeast VI. ATPase activity and protein synthesis in mitochondria isolated from nuclear mutants deficient in cytochromes

Abstract 1. The oligomycin-sensitive Mg2+-dependent ATPase activity of mitochondria isolated from wild-type yeast Saccharomyces cerevisiae was only slightly inhibited by atractyloside at concentrations which entirely prevented oxidative phosphorylation. This indicated that most of the ATPase in these mitochondrial preparations was located outside the atractyloside-sensitive barrier and did not participate in the energy-transfer process. 2. ATPase activity of mitochondria isolated from nuclear gene mutants deficient in a single cytochrome, a, b, or c, respectively, was strongly inhibited by oligomycin. The mitochondria from these mutants, like those from the wild-type strain, were able to incorporate amino acids into protein. 3. Mitochondrial ATPase activity of single nuclear gene mutants deficient in both cytochromes a and b was only slightly inhibited by oligomycin. These mitochondria were incapable of incorporating amino acids into protein. The mitochondria from these nuclear mutants thus resembled mitochondria of cytoplasmic respiration-deficient mutants. 4. The results suggest that mitochondrial cytochromes may be coded by nuclear genes and that product(s) of mitochondrial protein synthesis may be required for integrating the cytochromes a and b and the components of the oligomycin-sensitive ATPase complex into the mitochondrial membranes.

Genetic determination of the mitochondrial adenine nucleotide translocation system and ITS role in the eukaryotic cell

SummaryOn integrating experimental data published previously, the following picture of the mitochondrial adenine nucleotide(AdN) translocation system is being presented: 1.The AdN translocation system serves not only to transport ATP synthesized within mitochondria into the cytosol but also to transport cytosolic ATP into the mitochondria when oxidative phosphorylation is not functioning.2.The AdN translocator is coded for by nuclear genes and the mitochondrial protein synthesis is not involved in its formation.3.The AdN translocation system must be preserved and functioning even in cells which could dispense with oxidative phosphorylation. It assures appropriate concentrations of intramitochondrial ATP.4.The intramitochondrial ATP is required for normal replication of mitochondrial DNA. This supports the view that the mitochondrion is a self-replicating semi-autonomous organelle.5.The appropriate concentration of ATP must be present in mitochondria to make possible cell growth or multiplication. This points to a direct or indirect role of mitochondria in the control of cell proliferation.

Solubility of the intramitochondrially synthesized protein and other membrane proteins of rat liver mitochondria in acidic or neutral chloroform-methanol.

Abstract Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, v v /, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver in vivo is presented.

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria.

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.

Synthesis and intracellular transport of cytochrome oxidase subunit IV and ADP/ATP translocator protein in intact hepatoma cells

The biosynthesis of two mitochondrial membrane proteins - subunit IV of cytochrome oxidase and ADP/ATP translocator protein was studied in intact ascites hepatoma cells. Using pulse-chase labeling and rapid cell fractionation it was possible to identify the precursoric forms of these inner mitochondrial membrane proteins. It was found that the subunit IV of cytochrome oxidase is synthesized in the cytoplasm of mammalian cells in the form of a larger precursor while ADP/ATP translocator protein is synthesized in the form that is electrophoretically undistinguishable from the mature membrane integrated form.