Competition of spontaneous protein folding and mitochondrial import causes dual subcellular location of major adenylate kinase

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.     

Redundant mitochondrial targeting signals in yeast adenylate kinase

Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space. It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown. The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria. However, an Aky2 mutant lacking this sequence is imported with about the same efficiency as the wild type. To identify possible import-relevant information in the interior, parts of Aky2p were exchanged by homologous in vitro recombination for the respective segments of the purely cytoplasmic isozyme, Ura6p. Import studies revealed an internal region of about 40 amino acids, which was sufficient to direct the chimera to mitochondria but not for correct submitochondrial sorting. The respective Ura6p hybrid was arrested in the mitochondrial membrane at a position where it was inaccessible to protease but was released by alkaline extraction, suggesting that it had entered an import channel and passed the initial steps of recognition and uptake. Site-specific mutations within the presumptive address-specifying segment identified the amphipathic helix 5. A Ura6 mutant protein in which helix 5 had been replaced with the respective sequence from Aky2p was imported, and this address sequence cooperates with the N terminus in the respective double mutant in a synergistic fashion.     

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis.

Site-directed mutagenesis and deletions were used to study mitochondrial import of a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence 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 isozymes 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 displayed increased or, in the case of a deletion of the 8 N-terminal triplets, decreased import efficiencies. Although the N-terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

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

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 mitochondrial 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.     

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.     

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins.

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.     

The role of adenylate kinase in dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

To investigate the existence of dynamic adenine nucleotide (AdN) compartment in the mitochondrial intermembrane space, we used reconstituted systems consisting of (i) functional intact liver and heart mitochondria and (ii) pyruvate kinase plus phosphoenolpyruvate, both competing for ADP either formed in the intermembrane space by adenylate kinase or added directly into, or regenerated by ATPase within, the extramitochondrial space. It is shown that ADP formation in the mitochondrial intermembrane space is a prerequisite for a dominating oxidative phosphorylation in reconstituted systems, suggesting dynamic ADP compartmentation in that space.     

Oxidative folding competes with mitochondrial import of the small Tim proteins

All small Tim proteins of the mitochondrial intermembrane space contain two conserved CX(3)C motifs, which form two intramolecular disulfide bonds essential for function, but only the cysteine-reduced, but not oxidized, proteins can be imported into mitochondria. We have shown that Tim10 can be oxidized by glutathione under cytosolic concentrations. However, it was unknown whether oxidative folding of other small Tims can occur under similar conditions and whether oxidative folding competes kinetically with mitochondrial import. In the present study, the effect of glutathione on the cysteine-redox state of Tim9 was investigated, and the standard redox potential of Tim9 was determined to be approx. -0.31 V at pH 7.4 and 25 degrees C with both the wild-type and Tim9F43W mutant proteins, using reverse-phase HPLC and fluorescence approaches. The results show that reduced Tim9 can be oxidized by glutathione under cytosolic concentrations. Next, we studied the rate of mitochondrial import and oxidative folding of Tim9 under identical conditions. The rate of import was approx. 3-fold slower than that of oxidative folding of Tim9, resulting in approx. 20% of the precursor protein being imported into an excess amount of mitochondria. A similar correlation between import and oxidative folding was obtained for Tim10. Therefore we conclude that oxidative folding and mitochondrial import are kinetically competitive processes. The efficiency of mitochondrial import of the small Tim proteins is controlled, at least partially in vitro, by the rate of oxidative folding, suggesting that a cofactor is required to stabilize the cysteine residues of the precursors from oxidation in vivo.     

The role of Hot13p and redox chemistry in the mitochondrial TIM22 import pathway

The small Tim proteins in the mitochondrial intermembrane space participate in the TIM22 import pathway for assembly of the inner membrane. Assembly of the small TIM complexes requires the conserved "twin CX3C" motif that forms juxtapositional intramolecular disulfide bonds. Here we identify a new intermembrane space protein, Hot13p, as the first component of a pathway that mediates assembly of the small TIM complexes. The small Tim proteins require Hot13p for assembly into a 70-kDa complex in the intermembrane space. Once assembled the small TIM complexes escort hydrophobic inner membrane proteins en route to the TIM22 complex. The mechanism by which the small Tim proteins bind and release substrate is not understood, and we investigated the affect of oxidant/reductant treatment on the TIM22 import pathway. With in organello import studies, oxidizing agents arrest the ADP/ATP carrier (AAC) bound to the Tim9p-Tim10p complex in the intermembrane space; this productive intermediate can be chased into the inner membrane upon subsequent treatment with reductant. Moreover, AAC import is markedly decreased by oxidant treatment in Deltahot13 mitochondria and improved when Hot13p is overexpressed, suggesting Hot13p may function to remodel the small TIM complexes during import. Together these results suggest that the small TIM complexes have a specialized assembly pathway in the intermembrane space and that the local redox state of the TIM complexes may mediate translocation of inner membrane proteins.     

A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria

Summary 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.Abbreviations Ap5A P¹,P⁵-di(adenosine-5)pentaphosphate - adenylate kinase ATP: AMP phosphotransferase (EC 2.7.4.3) - Pak3p (Aky2p) protein product of the PAK3 (AKY2) gene - DHFR mouse dihydrofolate reductase     

Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis

The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2. The resultant isaDelta mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced. An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding. As predicted, Isa1p is targeted to the mitochondrial matrix. However, Isa2p is present within the intermembrane space of the mitochondria. Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed.     

Common Players in Mitochondria Biogenesis and Neuronal Protection Against Stress-Induced Apoptosis

Mitochondria biogenesis is a fundamental process for the organization and normal function of all cells. Since the majority of mitochondrial proteins are synthesized in the cytosol, protein import is the major mechanism for mitochondria biogenesis. We describe the different pathways that ensure correct targeting and intra mitochondrial sorting of mitochondrial proteins. The import process of several proteins of the mitochondrial intermembrane space relies on the Mitochondrial Import and Assembly 40 and Essential for respiration and vegetative growth 1 (Erv1) proteins that together constitute the oxidative folding machinery of the mitochondrial intermembrane space. Recent work has implicated the FAD-oxidase protein Erv1 (ad its human homolog Augmenter of Liver Regeneration) as an anti-apoptotic factor in mammalian cells (including neuronal cells) that undergo Reactive Oxygen Species-triggered apoptosis. The different roles of this protein as a key factor in mitochondria biogenesis, iron-sulfur cluster biogenesis and in neuronal protection against apoptosis are discussed.     

Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial intermembrane space

The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

How membrane proteins travel across the mitochondrial intermembrane space.

A newly discovered family of small proteins in the yeast mitochondrial intermembrane space mediates import of hydrophobic proteins from the cytoplasm into the inner membrane. Loss of one of these chaperone-like proteins from human mitochondria results in a disease that causes deafness, muscle weakness and blindness.     

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.     

Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP.

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.     

Functional and mutational characterization of human MIA40 acting during import into the mitochondrial intermembrane space

A first component involved in import into the mitochondrial intermembrane space, named Mia40, has been described recently in yeast. Here, we identified the human MIA40 as a novel and ubiquitously expressed component of human mitochondria. It belongs to a novel protein family whose members share six highly conserved cysteine residues constituting a -CXC-CX9C-CX9C- motif. Human MIA40 is significantly smaller than the fungal protein and lacks the N-terminal extension including a transmembrane region and mitochondrial targeting signal. It forms soluble complexes within the intermembrane space of human mitochondria. Depletion of MIA40 in human cells by RNA interference specifically affected steady-state levels of small and cysteine-containing intermembrane space proteins like DDP1 and TIM10A, suggesting that MIA40 acts along the import pathway into the intermembrane space. Studies on the in vivo redox state of human MIA40 demonstrated that it contains intramolecular disulfide bonds. Thiol-trapping assays revealed the co-existence of different oxidation states of human MIA40 within the cell. Furthermore, we show that the twin -CX9C- motif is specifically required for import and stability of MIA40 in mitochondria. Partial mutation of this motif affects stable accumulation of MIA40 in the intermembrane space, whereas mutation of all cysteine residues in this motif inhibits import in mitochondria. Taken together, we conclude that the biogenesis and function of MIA40 in the mitochondrial intermembrane space is dependent on redox processes involving conserved cysteine residues.     

The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.