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...

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinlanderin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber Mitochondria from various plant tissues reduce added oxaloacetate at high rates at the expense of NADH generated in the mitochondrial matrix. The reduction of oxaloacetate is so efficient that upon the addition of oxaloacetate to mitochondria oxidizing NADH-linked substrates, such as glycine, respiration can be almost totally inhibited (4,7,12,17,33). These results led to the proposal that, in these plant mitochondria, a malateoxaloacetate shuttle may play a physiological role in a transfer of redox equivalents between the mitochondrial matrix and the cytosol. Since the equilibrium of the malate dehydrogenase reaction lies far towards malate formation ), under physiological conditions the cytosolic malate concentrations are expected to be at least two orders of magnitude higher than the concentrations of oxaloacetate. Therefore, the functioning of a malate-oxaloacetate shuttle under physiological conditions requires that the uptake of oxaloacetate is half-saturated at micromolar concentrations of the substrate and that it is not greatly inhibited by a more than 100-fold excess of malate and other dicarboxylates, which has been demonstrated (14,21,34). The ability to catalyze a malate-oxaloacetate shuttle is found in mito-

Yeast mitochondrial adenylate kinase (high molecular mass form, gene locus: AKY2) is encoded on chromosome IV of the same DNA strand as histone H2A-I. The nontranslated intergenic region spans 560 bp, the nontranscribed spacer can be estimated to comprise at most 300 bp. The TATA-box sequence is contained in a striking environment consisting of 20 alternating pyrimidines and purines. The AKY2 transcript is made constitutively: (i) the cellular mRNA concentration does not vary significantly with either growth conditions or elapse of the cell cycle; (ii) p-galactosidase activity is about constant in yeast cells grown on various carbon sources after transformation with AKY2-promoter/lacZ fusions; (iii) primer elongation analysis shows that utilization of 5 initiation sites is qualitatively and quantitatively independent of the growth conditions and the carbon source used; (iv) Western blot analysis and adenylate kinase activity measurements indicate the absence of post-transciptional controls as well.Mitochondrial adenylate kinase; Constitutive promoter; Histone H2AI intergenic region; Promoter/lacZ fusion; Nucleotide sequence;(Saccharomyces cerevisiae)