One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions

In the yeast Saccharomyces cerevisiae, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). 59% of the sequence of GlyRS2 is identical to that of GlyRS1. Others have proposed that GRS1 and GRS2 encode the cytoplasmic and mitochondrial enzymes, respectively. In this work, we show that GRS1 encodes both functions, whereas GRS2 is dispensable. In addition, both cytoplasmic and mitochondrial phenotypes of the knockout allele of GRS1 in S. cerevisiae are complemented by the expression of the only known gene for glycyl-tRNA synthetase in Schizosaccharomyces pombe. Thus, a single gene for glycyl-tRNA synthetase likely encodes both cytoplasmic and mitochondrial activities in most or all yeast. Phylogenetic analysis shows that GlyRS2 is a predecessor of all yeast GlyRS homologues. Thus, GRS1 appears to be the result of a duplication of GRS2, which itself is pseudogene-like.     

Electrophoretic assay of folylpolyglutamate synthetase activity

A method is described for the determination of the activity of folylpolyglutamate synthetase based upon incorporation of reaction products into a covalent, ternary complex with tritiated 5-fluoro-2′-deoxyuridylate and thymodylate synthetase followed by electrophoretic identification of the enzyme-bound polyglutamate species.     

Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

The complete amino acid sequence of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae

The crystallizable cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63 000). Its primary structure was established using peptide sequences from four different digests of the native and citraconylated enzyme with trypsin, cyanogen bromide and staphylococcal protease. The oligonucleotide sequence of the structural gene was used as a template for the final alignment of the various peptides in the correct order.     

Relationship between cytoplasmic and mitochondrial apparatus of protein synthesis in yeast Saccharomyces cerevisiae

A conditional respiratory deficiency in yeast Saccharomyces cerevisiae is expressed as a result of a nuclear mutation in sup1 and sup2 genes (II and IV chromosomes, respectively), coding for a component of cytoplasmic ribosomes (Ter-Avanesyan et al. 1982). One such strain is studied here in detail. The strain is temperature-dependent and expresses a respiratory deficient phenotype at 20 degrees C but not at 30 degrees C. Moreover, the strain is simultaneously chloramphenicol-dependent and is able to grow on media containing glycerol or ethanol as a sole carbon source only in the presence of the drug. Chloramphenicol has a differential effect on protein synthesis in mitochondria of the parent strain and the mutant. Since chloramphenicol is a ribosome-targeting antibiotic we suggest that the differential effect of the drug on parent and mutant mitochondrial protein synthesis is due to the altered properties of mito-ribosomes of the mutant compared to those of the parent strain. Mitochondria of the mutant synthesize all the mitochondrially encoded polypeptides, however, in significantly lowered amounts. A suggestion is put forward for the existence of a common component (a ribosomal protein) for mito and cyto-ribosomes.     

The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.     

Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.     

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.     

Comparative electrophoretic study on proteins from mitochondrial and cytoplasmic ribosomes of Saccharomyces cerevisiae

By sodium dodecyl sulfate-acrylamide gel electrophoresis, proteinsof yeast mitochondrial ribosome were shown to be different fromthose of the cytoplasmic counterpart. Fourteen bands found withmitochondrial ribosome did not show any correspondence withthose of the cytoplasmic type. Further, relative amounts ofproteins with their molecular weight more than 50,000 were muchgreater in mitochondrial ribosome, while proteins with theirmolecular weight less than 27,000 were abundant in cytoplasmictype. (Received January 16, 1975; )     

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.     

CDC64 encodes cytoplasmic alanyl-tRNA synthetase, Ala1p, of Saccharomyces cerevisiae

The cdc64-1 mutation causes G(1) arrest in Saccharomyces cerevisiae corresponding to a type II Start phenotype. We report that CDC64 encodes Ala1p, an alanyl-tRNA synthetase. Thus, cdc64-1 might affect charging of tRNA(Ala) and thereby initiation of cell division.     

Transfer RNA genes in the mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Hybridization saturation analyses of mitochondrial DNA from 11 petite clones genetically characterized with respect to chloramphenicol and erythromycin resistance markers, have been carried out with 11 individual mitochrondrial transfer RNAs. Mitochondrial tRNA cistrons were lost, retained, or amplified in different petite strains. In some cases hybridization levels corrected for kinetic complexity of the mtDNA³ were two- to threefold greater than that for grande mtDNA indicating selective amplification, or increased number of copies, of the segment of mtDNA containing that tRNA cistron. Hybridization levels corrected for reduced kinetic complexity of petite mtDNAs in many cases were only 1 to 10% of that for grande mtDNA suggesting a low level of intracellular molecular heterogeneity of mtDNA with respect to tRNA cistrons. Some petite clones that retained tRNA genes continued to transcribe mitochondrial tRNAs, since tRNA isolated from these strains could be aminoacylated with Escherichia, coli synthetases and hybridized with mtDNA. Hybridization data allow us to order several of the tRNA cistrons on the mitochondrial genome with respect to the chloramphenicol and erythromycin antibiotic resistance markers.     

Fatty acid synthetase of Saccharomyces cerevisiae

A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein.     

The size of mitochondrial DNA from a cytoplasmic petite mutant of Saccharomyces cerevisiae

Summary Mitochondrial DNA has been isolated from a cytoplasmic petite mutant of Saccharomyces cerevisiae which has retained only about 2% of the mitochondrial wild type genome. The denatured DNA was analyzed by agarose gel electrophoresis and a homogeneous, single band of DNA was found. Petite and wild type mitochondrial DNAs exhibited similar gel electrophoretic mobilities. Using denatured DNA from the E. coli phages T4 and T3 for comparison a molecular weight of 55×10⁶ daltons has been calculated for the double-stranded petite mitochondrial DNA. On the basis of this observation most of the mitochondrial DNA of this petite mutant appeared to consist of a polymer of about 50 repeats to account for a size similar to that of the wild type molecule. Thus a regulatory mechanism might exist which keeps constant the physical size of the mitochondrial DNA molecule in spite of the elimination of large fractions of the wild type genome.Dedicated to Dr. Dr. h. c. Peter Michaelis on the occasion of his 75th birthday     

Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Comparison of amino acid compositions of mitochondrial and cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

Summary The amino-acid compositions of the mitochondrial ribosomal subunits of Saccharomyces cerevisiae have been determined and compared to those of cytoplasmic ribosomal subunits. For the large subunits, the mitochondrial and cytoplasmic ribosomes showed major differences in the proportions of arginine, alanine and methionine. For the small subunits, arginine, aspartic acid, alanine, valine and methionine showed marked differences.We have compared these amino-acid compositions with those already published of bacterial and eukaryotic ribosomes by a statistical method of data analysis. It appeared clearly that the yeast mitoribosomes are more distant from bacterial ribosomes than from eukaryotic cytoribosomes.Abbreviations r-proteins ribosomal proteins     

Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.