Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA^Lys _CUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA^Lys _CUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.     

Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast

It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs. In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria. This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur. To test whether this interaction is sufficient to drive import, we co-expressed A. thaliana tRNA(Ala)(UGC) and the precursor to the A. thaliana mitochondrial AlaRS in Saccharomyces cerevisiae. The A. thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo. However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A. thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors. We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants.     

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

Cytoplasmic tRNA(Lys)CUU is the only nuclear-encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl-tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl-tRNA synthetase is able to form specific and stable RNP complexes with the amino-acylated tRNA. Furthermore, an N-terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).     

Factors beyond enolase 2 and mitochondrial lysyl-tRNA synthetase precursor are required for tRNA import into yeast mitochondria

In yeast, the import of tRNA^Lys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

tRNA import into yeast mitochondria is regulated by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, one of two cytosolic lysine-tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre-Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Different internal metabolites trigger the induction of glycolytic gene expression in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the sugar-induced expression of various genes coding for glycolytic enzymes is triggered by increases in the concentrations of different internal metabolites. Here, we show that the induction of the glycolytic isoenzyme enolase 2 is strictly dependent on the abilities of different mutant strains to increase the level of glucose-6-phosphate after the addition of sugars. In contrast, the induction of alcohol dehydrogenase I is dependent on increasing concentrations of metabolites in the late stages of glycolysis.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.     

Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing

The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution."     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.     

Metabolic effects of mislocalized mitochondrial and peroxisomal citrate synthases in yeast Saccharomyces cerevisiae

Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively. A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle. To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria. A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle. Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p. We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p. Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes. Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes.     

Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.     

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.     

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.