In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: a paradigm of incipient evolution

Summary We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts caught in the act of speciation.     

ACR1, a gene encoding a protein related to mitochondrial carriers,is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

A genomic survey shows that the haloarchaeal type tyrosyl tRNA synthetase is not a synapomorphy of opisthokonts

The haloarchaeal-type tyrosyl tRNA synthetase (tyrRS) have previously been proposed to be a molecular synapomorphy of the opisthokonts. To re-evaluate this we have performed a taxon-wide genomic survey of tyrRS in eukaryotes and prokaryotes. Our phylogenetic trees group eukaryotes with archaea, with all opisthokonts sharing the haloarchaeal-type tyrRS. However, this type of tyrRS is not exclusive to opisthokonts, since it also encoded by two amoebozoans. Whether this is a consequence of lateral gene transfer or lineage sorting remains unsolved, but in any case haloarchaeal-type tyrRS is not a synapomorphy of opisthokonts. This demonstrates that molecular markers should be re-evaluated once a better taxon sampling becomes available.     

Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.

DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences.     

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.     

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.     

In vitro resolution of adeno-associated virus DNA hairpin termini by wild-type Rep protein is inhibited by a dominant-negative mutant of rep.

An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo. We expressed both wild-type (Rep78) and mutant (Rep78NTP) proteins in two helper-free expression systems consisting of either recombinant baculoviruses in insect cells or the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cell transient transfections. We analyzed nuclear extracts from both expression systems for the ability to complement uninfected HeLa cell cytoplasmic extracts in an in vitro terminal resolution assay in which a covalently closed AAV terminal hairpin structure is converted to an extended linear duplex. Although both Rep78 and Rep78NTP bound to AAV terminal hairpin DNA in vitro, Rep78 but not Rep78NTP complemented the terminal resolution assay. Furthermore, Rep78NTP was trans dominant for AAV terminal resolution in vitro. We propose that the dominant-negative replication phenotype of AAV genomes carrying the K340H mutation is mediated by mutant Rep proteins binding to the terminal repeat hairpin.     

The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.     

hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function.

Analysis of the cloned gene confirms that hsp26 of Saccharomyces cerevisiae is a member of the small heat shock protein superfamily. Previous mutational analysis failed to demonstrate any function for the protein. Further experiments presented here demonstrate that hsp26 has no obvious regulatory role and no major effect on thermotolerance. It is possible that the small heat shock protein genes originated as primitive viral or selfish DNA elements.     

Caffeine sensitivity of the yeast Saccharomyces cerevisiae MCD4 mutant is related to alteration of calcium homeostasis and degradation of misfolded proteins

The MCD4 gene of Saccharomyces cerevisiae encodes a protein involved in glycosylphosphatidylinositol (GPI) biosynthesis. However, some mutations in the MCD4 gene have pleiotropic effects that may not be related to the defects in GPI anchor synthesis. The ssu21 mutation in the MCD4 gene studied here causes sensitivity to caffeine. The screen for multicopy suppressors of caffeine sensitivity caused by the ssu21 mutation revealed genes involved in aminoglycerophospholipid metabolism, unfolded protein response, and protein degradation. The suppressor effect of all isolated genes was enhanced by an increase in concentration of external calcium, which is consistent with the ability of caffeine to block calcium entry into the yeast cell. Obtained results indicate that caffeine sensitivity caused by the ssu21 mutation could be due to cytoplasmic accumulation of misfolded proteins destined for degradation.     

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.     

A mutant precursor protein is poorly targeted to mitochondria and interferes in vivo with the import of other mitochondrial polypeptides inSaccharomyces cerevisiae

Cytoplasmically synthesized mitochondrial proteins are targeted to the organelle by an N-terminal presequence. We have identified a mutant ATP synthase-subunit with an altered presequence that results in a significant impairment of the transport of this polypeptide into mitochondria in vitro. When this mutant protein is expressed in yeast, its slow passage through the transport pathway interferes with the transport of a number of other mitochondrial proteins, indicating that they may share at least one common step in their transport into the mitochondrion.     

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.     

In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae were isolated which supported three mutations: two unlinked chromosomic mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial mutation conferring paromomycin sensitivity. When studied on isolated mitochondria, these mutants exhibited low phosphorylation efficiency and great proton permeability of their inner mitochondrial membrane. Experiments were carried out on whole cells: determination of growth rates, cellular yields and cellular respiration, either in the presence of triethyltin, an ATP synthase inhibitor, or in the presence of uncoupler, demonstrating that the proton leakage is actually a physiological phenomenon linked to the cold-sensitive phenotype. Experiments performed on isolated mitochondria confirmed the existence of such a proton leakage.     

Cloning and characterization of DIP1, a novel protein that is related to the Id family of proteins

Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1. This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle. However, the level of this protein increases severalfold during the differentiation of HL60 cells. The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle. DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins. The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer. These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined.