Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.     

COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.     

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene.

The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

The polyanion-binding domain of cytoplasmic Lys-tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability.

Cytoplasmic Lys-tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa. By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity. Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties. KRS1 is the structural gene for yeast cytoplasmic LysRS. It encodes a polypeptide with an amino-terminal extension composed of about 60-70 amino acid residues, compared to its prokaryotic counterpart. This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme. The aim of the present study was to probe in vivo the significance of this amino-terminal extension. We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino-terminal residues and, by site-directed mutagenesis, we have changed four or eight lysine residues from the amino-terminal segment of LysRS into glutamic acids. Engineered proteins were expressed in vivo after replacement of the wild-type KRS1 allele. The mutant enzymes displayed reduced specific activities (2-100-fold). A series of carboxy-terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino-terminal extensions. The removal of three residues led to a 2-7-fold increase in the specific activity of the mutant enzymes. This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme. All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly. The expression, as single-copy genes, of mutant enzymes with a complete deletion of the amino-terminal extension or with four Lys----Glu mutations, that displayed specific activities close to that of the wild-type LysRS, had no discernable effect on cell growth. We conclude that the polycationic extensions of eukaryotic aminoacyl-tRNA synthetases are dispensable, in vivo, for aminoacylation activities. The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide-chain extensions that characterize most, if not all, eukaryotic aminoacyl-tRNA synthetases.