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...

Sorting of cytoplasmically synthesized proteins to their target compartments usually is highly efficient so that cytoplasmic precursor pools are negligible and a particular gene product occurs at one subcellular location only. Yeast major adenylate kinase (Adk1p/Aky2p) is one prominent exception to this rule. In contrast to most mitochondrial proteins, only a minor fraction (6–8%) is taken up into the mitochondrial intermembrane space, whereas the bulk of the protein remains in the cytosol in sequence-identical form. We demonstrate that Adk1p/Aky2p uses a novel mechanism for subcellular partitioning between cytoplasm and mitochondria, which is based on competition between spontaneous protein folding and mitochondrial targeting and import. Folding is spontaneous and rapid and can dispense with molecular chaperons. After denaturation, enzymatic activity of Adk1p/Aky2p returns within a few minutes and, once folded, the protein is thermally and proteolytically very stable. In an uncoupled cell-free organellar import system, uptake of Adk1p/Aky2p is negligible, but can be improved by previous chaotropic denaturation. Import ensues independently of Hsp70 or membrane potential. Thus, nascent Adk1p/Aky2p has two options: either it is synthesized to completion and folds into an enzymatically active import-incompetent conformation that remains in the cytosol; or, during synthesis and before commencement of significant tertiary structure formation, it reaches a mitochondrial surface receptor and is internalized.

The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a ؉1 frameshift into the 3-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a ؉1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.Adenylate kinases constitute a family of highly conserved soluble proteins that catalyzes the interconversion of nucleoside phosphates (Noda, 1973) and, thus, fulfills an essential function in maintaining the energy charge in cells (Atkinson, 1977). In mammals three types of isozymes exist. They can be divided into short and long isoforms of 21 and 25 kDa molecular mass, respectively. The short form enzyme, AK, 1 resides in the cytoplasm. Based on differences in primary structure, substrate utilization, and subcellular location, two distinct subgroups of the 25-kDa form can be discriminated, called AK2 and AK3 (for a review, see Schulz (1987)). AK2 is located mainly in the mitochondrial intermembrane space and uses ATP⅐Mg 2ϩ as donor of the high energy phosphate, while AK3, which occurs in the mitochondrial matrix, uses GTP⅐Mg 2ϩ and ITP⅐Mg 2ϩ (Tomasselli et al., 1979a(Tomasselli et al., , 1986). The latter is thought to play a role in the interconversion of non-ATP nucleoside triphosphates, ITP, and GTP (Tomasselli et al., 1979b), generated by substrate chain phosphorylation through succinic thiokinase in the tricarboxylic acid cycle.By contrast, procaryotes were shown to contain only a single member of the adenylate kinase family, a long form isozyme (Brune et al., 1985). In yeast, the major isoform of adenylate kinases, Aky2p, is a protein of 24 kDa molecular mass displaying highest homology to mammalian AK2 isozymes. As in bacteria, this protein was considered to be the only adenylate kinase in ...

Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8%) and in the cytoplasm (approx. 90%). To identify the signal(s) on Aky2p that might be required for mitochondrial import, the N-terminal region was examined. The N-terminus of Aky2p can guide at least two cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information. In contrast, deletion of the eight N-terminal amino acid residues or the introduction of two compensating frameshifts into this segment does not abolish translocation into the organelle's intermembrane space. Thus internal targeting and sorting information must be present in Aky2p as well. Neither a pronounced amphiphilic alpha-helical moment nor positive charges in the N-terminal region is a necessary prerequisite for Aky2p to reach the intermembrane space. Even a surplus of negative charges in mutant N-termini does not impede basal import into the correct submitochondrial compartment. The potential to form an amphipathic alpha-helical structure of five to eight residues close to the N-terminus significantly improves import efficiency, whereas extension of this amphipathic structure, e.g. by replacing it with the homologous segment of Aky3p, a mitochondrial matrix protein from yeast, leads to misdirection of the chimaera to the matrix compartment. This shows that the topogenic N-terminal signal of Aky3p is dominant over the presumptive internal intermembrane space-targeting signal of Aky2p and argues that the sorting of wild-type Aky2p to the intermembrane space is not due to the presence in the protein of a specific sorting sequence for the intermembrane space, but rather is the consequence of being imported but not being sorted to the inner compartment. Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding. They are nevertheless efficiently rescued by uptake into mitochondria, suggesting a negative correlation between folding velocity (or folding stability) and efficiency of import.

Site-directed mutagenesis and deletions were used to study mitochondrial import of a major yeast adenylate kinase, AkyZp. This enzyme lacks a cleavable prescquence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface-exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase lsozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long-form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N-or C-terminal parts frequently disptayed increased or, in the case of a deletion ofthe 8 N-terminal triplets, decreased import cfhciencies. Although the N-terminus is important for targeting adenylatc kinase to mitochondria, other parameters like internal squsnce determinants and folding velocity of the nascent protein may also play a role, S~ccAurorrtycr ccrrvisiuc: ATP:AMP phosphotransferase; in vitro mutagenesis; mitochondrial import