Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.     

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.     

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.     

Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Purification and properties of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from L1210 cells

Serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase have been purified to near homogeneity from L1210 cells. Kinetic constants (Km and kcat) have been determined for both folate and non-folate substrates. The effect of increasing glutamate chain length on affinity and catalytic efficiency were determined for the four activities. The studies show that the structural and catalytic properties of the two L1210 enzymes are very similar to the corresponding enzymes purified from rabbit liver. Antibodies to both rabbit serine hydroxymethyltransferase and C1-tetrahydrofolate synthase cross-react with the corresponding L1210 enzymes. The intracellular concentration of active sites of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in L1210 cells are both 9 microM. The combined concentration of these two enzymes exceeds the previously reported concentration of 10 microM for total intracellular folates. A network thermodynamic computer model of one carbon metabolism (Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016-17023) suggests that complete inhibition of cytosolic serine hydroxymethyltransferase would neither significantly decrease the rates of biosynthesis of purines and thymidylate nor significantly alter the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate with subsequent inhibition of dihydrofolate reductase.     

Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity. During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.     

Purification, immunoassay, and tissue distribution of rat C1-tetrahydrofolate synthase

C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.     

Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae

Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae. The enzyme was purified 472-fold to apparent homogeneity. The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P. The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+. Yeast MAP failed to cleave methionine p-nitroanilide. Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser. MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g. Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg. His, Leu, Met, or Tyr). Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Werlen, R. C., Garber, P., and Movva, N. R. (1987) Proc. Natl, Acad. Sci. U.S.A. 84, 2718-2722). In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S. M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984).     

Purification and characteristics of a mitochondrial endonuclease from the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.     

Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was purified 12 000-fold to homogeneity from yeast by a three-step procedure including acid precipitation, anion-exchange chromatography, and guanosine 5' -monophosphate affinity chromatography. The enzyme is a dimer consisting of two, probably identical, subunits of Mr 29 500. The enzyme recognized hypoxanthine and guanine, but not adenine or xanthine, as substrates. An antiserum against both native and denatured enzyme has been raised and shown to be specific for the enzyme. The antiserum has no affinity for Chinese hamster or human HPRT but does recognize subunits of yeast HPRT as well as some cyanogen bromide fragments of the enzyme.     

Stabilization and characterization of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae

Histidine-tagged homocitrate synthase from Saccharomyces cerevisiae was purified to about 98% using a Ni-NTA resin and stabilized using a combination of 100 mM guanidine hydrochloride, 100 mM alpha-cyclodextrin, and 600 mM ammonium sulfate. The enzyme was assayed using dichlorophenol indophenol (DCPIP) as an oxidant to oxidize the CoASH produced in the reaction. A stoichiometry of 1:1 was obtained between DCPIP and CoASH. Kinetic parameters for the stable enzyme at pH 7.5 are: Km (AcCoA), 24 microM: Km (alpha-kg), 1.3 mM; and kcat, 37 min(-1). The enzyme, in the absence of reactants, self-associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed tryptophan residue and a mixed (alpha/beta) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide suggest that the microenvironment around the single tryptophan residue of the enzyme has some positive charge.     

Purification and characterization of the indole-3-glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae

The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity. Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.     

Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase

Membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VAL2C(YEpCHO1). VAL2C(YEpCHO1) contains a hybrid plasmid bearing the structural gene for phosphatidylserine synthase and overproduces the enzyme 6-7 fold (Letts, V. A., Klig, L. S., Bae-Lee, M., Carman, G. M., and Henry, S. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7279-7283) compared to wild-type S288C. The purification procedure included Triton X-100 extraction of the microsomal membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and DE-53 chromatography. The procedure yielded a preparation from each strain containing a major peptide band (Mr = 23,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylserine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0. The apparent Km values for serine and CDP-diacylglycerol were 0.58 mM and 60 microM, respectively. Thioreactive agents inhibited enzyme activity. The enzyme was thermally labile above 40 degrees C. Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.     

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.