No abstract

Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeast is targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disrupted yeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.

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