Macromolecular Synthesis and Energy Level in a Mitochondrial Conditional Yeast Mutant, tsm-8

Bandlow, Wolfhard; Metzke, Roland; Klein, Albrecht; Kotzias, Klaus; Doxiadis, Ilias; Bechmann, Helga; Schweyen, Rudolf J.; Kaudewitz, F.

doi:10.1111/j.1432-1033.1977.tb11605.x

Cited by 5 publications

(1 citation statement)

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“…RNA was isolated from cells disrupted by shaking (by hand) in the presence of glass beads [27] and 4 M guanidinium isothiocyanate followed by centrifugation (16 h, 135000 x g) through a cushion of CsCl (nyo = 1.408). Respiration of whole cells was measured polarographically as described [28]. Protein was determined using the bicinchoninic acid reagent (Atlanta, Pierce Biochemicals, Heidelberg) according to the instructions of the manufacturer.…”

Section: Electroblotting Immune Decoration and Miscellaneous Proceduresmentioning

confidence: 99%

Yeast adenylate kinase is active simultaneously in mitochondria and cytoplasm and is required for non‐fermentative growth

Bandlow

Strobel

Zoglowek

et al. 1988

European Journal of Biochemistry

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Displacement of the single copy structural gene for yeast adenylate kinase (long version) by a disrupted nonfunctional allele is tolerated in haploid cells. Since adenylate kinase activity is a pre-requisite for cell viability, the survival of haploid disruption mutants is indicative of the presence of an adenylate kinase isozyme in yeast, capable of forming ADP from AMP and, thus, of complementing the disrupted allele. The phenotype of these disruption mutants is pet, showing that complementation occurs only under fermentative conditions. Even on glucose, growth of the disruption mutants is slow. Adenylate kinase activity is found both in mitochondria and cytoplasm of wild type yeast. The disruption completely destroys the activity in mitochondria, whereas in the cytoplasmic fraction about 10% is retained. An antibody raised against yeast mitochondria1 adenylate kinase recognizes cross-reacting material both in mitochondria and cytoplasm of the wild type, but fails to do so in each of the respective mutant fractions. The data indicate that yeast adenylate kinase (long version, AKY2) simultaneously occurs and is active in mitochondria and cytoplasm of the wild type. Nevertheless, it lacks a cleavable pre-sequence for import into mitochondria. A second, minor isozyme, encoded by a separate gene, is present exclusively in the cytoplasm.Adenylate kinases are indispensable for cell viability, since they are required to activate AMP at the expense of ATP cleavage to yield two molecules of ADP. Two different types of the enzyme have been found in various organisms [l, 21. A low-molecular-mass form was isolated and characterized from the cytoplasm of cells from several vertebrate tissues [3 -61, whereas a high-molecular-mass version was found to be present in the mitochondria of these cells [7-lo], as well as in yeast [11 -141 and bacteria [15, 161. The latter two microorganisms have been reported to have only the long-form isozyme located in their cytoplasm.The amino acid [I31 and nucleotide [17] sequences of the yeast enzyme have been published recently and found to be highly similar to both the enzymes from Escherichiu coli [15] and from bovine heart mitochondria [lo]. They differ from the short cytoplasmic vertebrate isozyme mainly by the insertion of a block of 31 amino acids into the C-terminal third of the enzyme. The exclusive cytoplasmic location of the yeast enzyme [l 1 ~ 141 has been questioned by other groups. On the one hand, it was observed that yeast mitochondria contain an adenylate kinase activity of their own [18, 191 which is located in the intermembrane space [20]. On the other, it was concluded on the basis of the much higher similarity with the mitochondrial isoform from vertebrates and the presence of the conserved insertion of 31 amino acids that this protein was more likely to constitute the mitochondrial isozyme in yeast [I 71.Disruptions of the gene, combined with cellular fractionation and immunological and enzymatic assays, show that the long version of adenylate kinase occurs in an a...

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Section: Electroblotting Immune Decoration and Miscellaneous Proceduresmentioning

confidence: 99%

Yeast adenylate kinase is active simultaneously in mitochondria and cytoplasm and is required for non‐fermentative growth

Bandlow

Strobel

Zoglowek

et al. 1988

European Journal of Biochemistry

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On the formation of ρ−petites in yeast

et al. 1977

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1. In non-fermentable substrates growth of mutant tsm-8 cells of Saccharomyces cerevisiae is restricted to about one generation after shift from 23 to 35 degrees C. Non-permissive conditions (35 degrees C, glycerol) cause a gradual decrease in respiration to about 20% of the activity at permissive temperature 23 degrees C). 2. Anaerobically grown and glucose-repressed mutant cells exhibit a decreased adaptation rate of mitochondrial functions to aerobic growth and non-fermentative growth, even at 23 degrees C, as revealed by determination of respiratory rates and mitochondrial protein synthesis. 3. At 35 degrees C, rho+ cells of mutant tsm-8 are converted to p- cells within 6-8 generations of growth, in all fermentable substrates tested. Drugs or antibiotics as nalidixic acid, acriflavin, chloramphenicol and erythromycin, bongkrecic acid, antimycin and FCCP, as well as anaerobiosis, have little or no influence on this kinetics. A heat shock does not yield rho- petites to a significant extent. 4. Reversion of tsm-8 cells to wild type function, which occurs spontaneously with a frequency of 10(-8), is found to be due to a mitochondrial mutational event.

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