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.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.     

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.     

A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.     

Saccharomyces cerevisiae mitochondria lack a bacterial-type sec machinery.

The bacterial Sec genes encode a generalized protein export machinery. Although the mitochondria present in eukaryotic cells are derived from bacterial ancestors, a comprehensive search of the complete genomic sequence for the eukaryotic yeast Saccharomyces cerevisiae did not reveal any close homologs of the bacterial Sec genes, strongly suggesting that yeast mitochondria lack a generalized bacterial-type export system. This finding has implications for the sorting of imported mitochondrial proteins to the intermembrane space compartment, and also for the insertion of mitochondrially encoded proteins into the inner membrane.     

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae

The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.     

Mak10, a Glucose-Repressible Gene Necessary for Replication of a Dsrna Virus of Saccharomyces Cerevisiae, Has T Cell Receptor α-Subunit Motifs

The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae. We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10. This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions. We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A. Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed. Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed. The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression. These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.     

Identification and functions of new transporters in yeast mitochondria

The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.     

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.     

Accumulation and release of the osmolyte glycerol is independent of the putative MIP channel Spac977.17p in Schizosaccharomyces pombe

Schizosaccharomyces pombe accumulates glycerol as an osmotic regulatory solute in response to hyper-osmotic conditions. Upon a decrease in the external osmolarity, the intracellular glycerol levels should be adjusted in order to attain osmotic homeostasis. In this study, the patterns and kinetics of glycerol export from S. pombe were investigated. Upon a decrease in external osmolarity, glycerol was rapidly exported from cells to the external medium. The amount of glycerol released from the cells was proportional to the degree of change in the external osmolarity. The export process was well controlled and was not affected by reduced temperature. This points to S. pombe controlling glycerol export using specialized facilitating proteins as has been found in Saccharomyces cerevisiae where a MIP family channel protein Fps1p is involved. Analysis of the S. pombe databases revealed a putative transport protein (Spac977.17p) with homology to glycerol channel proteins of the MIP family. However, expression of the gene into the S. cerevisiae strain lacking a glycerol channel protein (fps1Delta mutant), did not complement the defect in glycerol export during hypo-osmotic stress. Deletion of spac977.17, did not affect glycerol accumulation or release in S. pombe. The patterns and kinetics of glycerol release in the mutant were similar to those of the wild type strains suggesting that the export process is independent of Spac977.17p, the only putative MIP family glycerol channel homologue in S. pombe. While the process of glycerol export in response to hypo-osmotic stress is similar to budding yeast, the underlying molecular mechanism in S. pombe appears distinct from that described in S. cerevisiae. Further studies are needed to elucidate the physiological role of the Spac977.17p channel.     

Zuotin, a DnaJ molecular chaperone, stimulates cap-independent translation in yeast

A small inhibitor RNA (IRNA) isolated from yeast has previously been shown to efficiently block poliovirus and hepatitis C virus IRES-mediated translation by sequestering mammalian RNA-binding (transacting) factors that play important roles in cap-independent translation. Here we have investigated the IRNA-binding proteins that might be involved in cap-independent translation in the yeast Saccharomyces cerevisiae. We have identified Zuotin, a DnaJ chaperone protein similar to mammalian HSP-40 chaperone, which interacts strongly with IRNA. Using ZUO1-deleted S. cerevisiae, we demonstrate a preferential requirement of Zuo1p for cap-independent translation mediated by the 5' untranslated region of the yeast TFIID mRNA. Further studies using zuo1delta S. cerevisiae complemented with various Zuo1p mutants indicate that the DnaJ domain of Zuo1p, known to influence its interaction with HSP-70, significantly affects cap-independent translation. These results demonstrate for the first time a role for an established chaperone protein in cap-independent translation of a cellular mRNA.     

Identification and comparative analysis of the large subunit mitochondrial ribosomal proteins of Neurospora crassa

The mitochondrial ribosome (mitoribosome) has highly evolved from its putative prokaryotic ancestor and varies considerably from one organism to another. To gain further insights into its structural and evolutionary characteristics, we have purified and identified individual mitochondrial ribosomal proteins of Neurospora crassa by mass spectrometry and compared them with those of the budding yeast Saccharomyces cerevisiae. Most of the mitochondrial ribosomal proteins of the two fungi are well conserved with each other, although the degree of conservation varies to a large extent. One of the N. crassa mitochondrial ribosomal proteins was found to be homologous to yeast Mhr1p that is involved in homologous DNA recombination and genome maintenance in yeast mitochondria.     

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation.

Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S. cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thus, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.     

Mitochondrial GFA2 is required for synergid cell death in Arabidopsis

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.     

Ionophores and intact cells. II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg2+ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Mutations Affecting a Yeast Mitochondrial Inner Membrane Protein, Pnt1p, Block Export of a Mitochondrially Synthesized Fusion Protein from the Matrix

The machinery that inserts mitochondrially encoded proteins into the inner membrane and translocates their hydrophilic domains through the membrane is poorly understood. We have developed a genetic screen for Saccharomyces cerevisiae mutants defective in this export process. The screen is based on the fact that the hydrophilic polypeptide Arg8(m)p is exported from the matrix if it is synthesized within mitochondria as a bifunctional Cox2p-Arg8(m)p fusion protein. Since export of Arg8(m)p causes an Arg(-) phenotype, defective mutants can be selected as Arg(+). Here we show that mutations in the nuclear gene PNT1 block the translocation of mitochondrially encoded fusion proteins across the inner membrane. Pnt1p is a mitochondrial integral inner membrane protein that appears to have two hydrophilic domains in the matrix, flanking a central hydrophobic hairpin-like anchor. While an S. cerevisiae pnt1 deletion mutant was more sensitive to H(2)O(2) than the wild type was, it was respiration competent and able to export wild-type Cox2p. However, deletion of the PNT1 orthologue from Kluyveromyces lactis, KlPNT1, caused a clear nonrespiratory phenotype, absence of cytochrome oxidase activity, and a defect in the assembly of KlCox2p that appears to be due to a block of C-tail export. Since PNT1 was previously described as a gene affecting resistance to the antibiotic pentamidine, our data support a mitochondrial target for this drug.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.