Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.     

cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.     

Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme

Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action. Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure. Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs. ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature. Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1. These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability. The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated.     

Multisite surface electromyography and complementary healing intervention: a comparative analysis

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form alpha alpha' beta 2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic alpha subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of alpha subunit described in other species, although, uniquely, Y. lipolytica CKII alpha lacks cysteines. We find that the alpha subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKII alpha expression and CKII alpha activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.     

Sequence of the fifth largest subunit of RNA polymerase II from plants

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.     

Cytokine synthesis of human monocytes stimulated by triple or single helical conformer of an antitumour (1-->3)-beta-D-glucan preparation, sonifilan

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

Mutational analysis of yeast mRNA capping enzyme

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.     

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.     

The subunit f of mitochondrial yeast ATP synthase--characterization of the protein and disruption of the structural gene ATP17

The subunit f of the yeast F1F0ATP synthase has been isolated from the purified enzyme. Amino acid composition, protein and peptide sequencing were performed. The data are in agreement with the sequence of the predicted product of the gene D9481.21 identified on the Saccharomyces cerevisiae chromosome IV. A 303-bp open reading frame encoding a 101-amino acid polypeptide is described. The deduced amino acid sequence from the ATP17 gene is 6 amino acids longer than the mature protein, which displays a molecular mass of 10567 Da. The protein is basic with a short hydrophobic segment located in the C-terminal part of the subunit. Subunit f remained associated with other F0 subunits upon sodium bromide treatment of the whole enzyme. A null mutant was constructed. The disrupted strain was unable to grow on glycerol medium and the mutation was recessive; rho- cells arose spontaneously. The null mutant mitochondria were devoid of oligomycin-sensitive ATPase, but still contained an active F1, while the subunits f, 6 and 8 were absent.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Purification of the pyruvate dehydrogenase multienzyme complex of Zymomonas mobilis and identification and sequence analysis of the corresponding genes

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1alpha (38 kDa) and E1beta (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAalpha (1,065 bp) and pdhAbeta (1,389 bp), encoding the E1alpha and E1beta subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAalpha, pdhAbeta, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1beta subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.     

Intact proinsulin, des 31,32 proinsulin, and specific insulin concentrations among nondiabetic and diabetic subjects in populations at varying risk of type 2 diabetes

Starting with crude yeast mitochondria, the intron homing endonuclease, I-SecIV, was purified to near homogeneity. This highly purified enzyme differs from some other well-characterized yeast mitochondrial intron-encoded endonucleases in terms of its structure and DNA cleavage specificity. The enzyme is a heterodimer with a native molecular mass of 92 kDa. A small catalytic subunit (32 kDa) is probably encoded largely or entirely by intron 5 alpha of the cytochrome oxidase subunit I gene. A larger polypeptide subunit (60 kDa) may be a nuclear factor necessary for intron mobility. I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates. Analysis of kinetic parameters shows that the purified enzyme has a very high affinity for DNA and exhibits low turnover which may have implications for subsequent steps in the intron homing process.     

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.     

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart

We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.     

Norepinephrine transporters in rat placenta labeled with [3H]nisoxetine

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.