Saccharomyces cerevisiae VIG9 Encodes GDP-mannose Pyrophosphorylase, Which Is Essential for Protein Glycosylation
1997; Elsevier BV; Volume: 272; Issue: 26 Linguagem: Inglês
10.1074/jbc.272.26.16308
ISSN1083-351X
AutoresHitoshi Hashimoto, Akira Sakakibara, Makari Yamasaki, Koji Yoda,
Tópico(s)Ubiquitin and proteasome pathways
ResumoA genomic DNA fragment that complements a newly identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae was cloned. Chromosomal integration of this fragment by homologous recombination indicated that it contains the wild type VIG9 gene. The nucleotide sequence was determined. A predicted gene product showed significant amino acid sequence homology with several bacterial enzymes that catalyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from sugar phosphates and (deoxy)ribonucleotide triphosphate. We examined the enzyme activity to synthesize GDP-mannose in the cell extracts of the wild type, vig9–1 mutant, and VIG9transformant yeasts. Reduction of the activity in the mutant cell and its restoration by VIG9 suggested that the VIG9gene is the structural gene for GDP-mannose pyrophosphorylase ofS. cerevisiae which catalyzes the production of GDP-mannose. We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli. A genomic DNA fragment that complements a newly identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae was cloned. Chromosomal integration of this fragment by homologous recombination indicated that it contains the wild type VIG9 gene. The nucleotide sequence was determined. A predicted gene product showed significant amino acid sequence homology with several bacterial enzymes that catalyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from sugar phosphates and (deoxy)ribonucleotide triphosphate. We examined the enzyme activity to synthesize GDP-mannose in the cell extracts of the wild type, vig9–1 mutant, and VIG9transformant yeasts. Reduction of the activity in the mutant cell and its restoration by VIG9 suggested that the VIG9gene is the structural gene for GDP-mannose pyrophosphorylase ofS. cerevisiae which catalyzes the production of GDP-mannose. We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli. Most secretory and membrane proteins of eukaryotic cells are modified by glycosylation. Glycosylation endows protein with a wide variety of characteristics that are important in their cellular functions. Glycosylation occurs in the secretory pathway, and three types of modification are known: (i) attachment of N-linked saccharides to asparagine residues; (ii) attachment ofO-linked saccharides to serine or threonine residues; and (iii) attachment of glycosylphosphatidylinositol anchors at the COOH termini. This is also the case with the yeast Saccharomyces cerevisiae, although mannose is the main component ofN- and O-linked saccharides, and varieties of sugars are found in those of higher eukaryotes. In yeasts, the fourth type of protein-saccharide linkage was suggested for the covalent linkage between the cell wall proteins and glucans, although details are yet to be studied (1Klis F.M. Yeast. 1994; 10: 851-869Crossref PubMed Scopus (487) Google Scholar). Several excellent reviews on glycosylation in yeasts have been published (2Ballou C.E. Methods Enzymol. 1990; 185: 440-470Crossref PubMed Scopus (275) Google Scholar, 3Herscovics H. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (441) Google Scholar, 4Kukuruzinska M.A. Bergh M.L.E. Jackson B.J. Annu. Rev. Microbiol. 1987; 56: 915-944Google Scholar, 5Lehle L. Tanner W. Montreuil J. Schachter H. Vliegenthart J.F.G. Glycoproteins: New Comprehensive Biochemistry. Elsevier Science, New York1995: 475-520Google Scholar). N-Linked saccharides are first transferred to proteins as a Glc3Man9GlcNAc2 unit from the Dol 1The abbreviations used are: Dol, dolichol-GDP-Man, guanidine diphosphomannose; GST, glutathioneS-transferase; ORF, open reading frame. -PP-intermediate when proteins are translocated into the endoplasmic reticulum. They are then trimmed to form the core N-linked saccharide, Man8GlcNAc2, which is common in eukaryotes. After proteins are transported from the endoplasmic reticulum to the Golgi apparatus in S. cerevisiae, core N-linked saccharides receive various quantities of mannose, depending on the protein species, one by one from GDP-Man. O-Linked saccharides that are composed exclusively of mannose in S. cerevisiae are also transferred to proteins in the Golgi apparatus sequentially from GDP-Man except for the first mannose, which is transferred from Man-P-Dol in the endoplasmic reticulum. As mannose is the major component of both N- and O-linked saccharides in S. cerevisiae, formation of GDP-Man, the activated form of mannose, should be vitally important. In S. cerevisiae, genetic approaches have been utilized in many scientific studies. To study the mechanism and function of glycosylation, mutants defective in glycosylation have been obtained in various ways. The mnn mutants were selected based on changes in cell surface characteristics such as binding of dyes, ion exchange matrix, or antisera (2Ballou C.E. Methods Enzymol. 1990; 185: 440-470Crossref PubMed Scopus (275) Google Scholar). [3H]Mannose suicide selection was used to obtain the alg mutants, which are defective in the formation of oligosaccharide-PP-Dol (6Huffaker T.C. Robbins P.W. J. Biol. Chem. 1982; 257: 3203-3210Abstract Full Text PDF PubMed Google Scholar), and the och1mutant (7Nakayama K. Nagasu T. Shimma Y. Kuromitsu J. Jigami Y. EMBO J. 1992; 11: 2511-2519Crossref PubMed Scopus (245) Google Scholar). Some of the sec mutants show the glycosylation-defective phenotype because glycosylation occurs during the process of secretion. Glycosylation mutants were also obtained as resistant or hypersensitive to chemicals or killer toxins; thevrg mutants were obtained by vanadate resistance (8Ballou L. Hitzeman R.A. Lewis M.S. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3209-3212Crossref PubMed Scopus (114) Google Scholar), thecwh mutants were obtained by Calcofluor White hypersensitivity (9Ram A.F.J. Wolters A. Hoopen R.T. Klis F.M. Yeast. 1994; 10: 1019-1030Crossref PubMed Scopus (273) Google Scholar), and the kre2 mutant was obtained by K1 killer toxin resistance (10Hill K. Boone C. Goebl M. Puccia R. Sdicu A.-M. Bussey H. Genetics. 1992; 130: 273-283Crossref PubMed Google Scholar). The erd1 mutant was obtained by a retention defect of the endoplasmic reticulum resident proteins (11Hardwick K.G. Lewis M.J. Semenza J. Dean N. Pelham H.R.B. EMBO J. 1990; 9: 623-630Crossref PubMed Scopus (121) Google Scholar). Studies of these mutants and the cloned wild type genes have been helpful in elucidating the biosynthetic enzymes and intermediate structures of oligosaccharides. To increase the collection of glycosylation-defective mutants, we screened for vanadate-resistant colonies as described previously but not screened exhaustively by Ballou et al. (8Ballou L. Hitzeman R.A. Lewis M.S. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3209-3212Crossref PubMed Scopus (114) Google Scholar). This selection is excellent because mutants are positively selected, although the biochemical rationale of enrichment is not clear. We isolated glycosylation-defective mutants of nine complementation groups. 2A. Sakakibara, H. Hashimoto, M. Yamasaki, and K. Yoda, in preparation. One of them, vig9(vanadate-resistant and immatureglycosylation), was found to be a novel mutation defective in the formation of GDP-Man. We report here the cloning and analysis ofVIG9, the structural gene for GDP-Man pyrophosphorylase ofS. cerevisiae. S. cerevisiae TM10 (MATa, leu2 his3 ura3 trp1), W303 (MATa/α, ura3/ura3 leu2/leu2 trp1/trp1 his3/his3 ade2/ade2 can1/can1), M9–2D (MATα, mnn9 ade2 lys2 leu2 trp1 his3 ura3), H17–6C (MATα, vig9–1 leu2 ura3 trp1), J12 (MATa, vig9–2 lys1), and H17W (MATa/α, vig9–1/vig9–1 ADE2/ade2 HIS3/his3 leu2/leu2 ura3/ura3) were used. Escherichia coli DH5α (F−, supE44 ΔlacU169 φ80lacZΔM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used in plasmid propagation, andE. coli BL21/pT-groE (F−, ompT hsdS gal dcm, pT-groE) was used in preparation of GST fusion proteins (12Yasukawa T. Kanei-Ishii C. Maekawa T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Yeast was usually grown at 30 °C in YEPD (1% Bacto yeast extract (Difco), 2% Bacto-peptone (Difco), and 2% glucose) medium or in SD (0.67% Bacto yeast nitrogen base without amino acids (Difco), 2% glucose, and appropriate supplements) medium. Invertase-inducing medium, YEP2S, contained 2% sucrose instead of glucose in YEPD. Solid media were supplemented with 2% Bacto-agar (Difco). Standard methods were used (13Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. 2nd ed. Academic Press, New York1991Google Scholar, 14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) unless otherwise stated. Enzymes were purchased from either Boehringer Mannheim or Takara Shuzo Co. pRS series plasmids (15Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) and pYES2.0 (Invitrogen) were used as vectors. The secretory invertase was analyzed according to the method of Gabriel and Wang (16Gabriel O. Wang S.-F. Anal. Biochem. 1969; 27: 545-554Crossref PubMed Scopus (188) Google Scholar). Cells were grown to mid-logarithmic phase in 1 ml of YEPD medium, centrifuged, washed with water, resuspended in 1 ml of YEP2S, and incubated further for 2 h. Cells were collected by centrifugation, washed once, and incubated in 100 μl of 10 mm Tris-HCl, pH 8.0, containing 0.9 m sorbitol, 0.1 m EDTA, 10 mm dithiothreitol, and 100 μg/ml Zymolyase 100T (Seikagaku Kogyo) at 37 °C for 30 min. The released periplasmic fraction was separated from spheroplasts by centrifugation. To analyze the native enzyme, the periplasmic fraction was subjected to 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The gel was bathed in 0.1 m sodium acetate, pH 5.1, containing 0.1 m sucrose at 37 °C for 1 h, washed with water, placed in 0.1% 2,3,5-triphenyltetrazolium chloride, 0.5m NaOH, and boiled to detect red bands. To analyze the denatured enzyme, the periplasmic proteins were labeled with biotin by adding d-biotinoyl-ε-aminocapronic acid-N-hydroxysuccinimide ester (100 μg/ml, Boehringer Mannheim), and the reaction was stopped by NH4Cl (50 mm). This solution was incubated at 4 °C for 1 h after adding anti-invertase antiserum and then 8 h after adding protein A-Sepharose (Pharmacia Biotech Inc.). Beads were washed with TBSN (50 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40) three times. The immunoprecipitated samples were analyzed by SDS-polyacrylamide gel electrophoresis. Proteins were blotted to a polyvinylidene difluoride membrane and detected with Streptoavidin-peroxidase. Endoglycosidase H (0.3 milliunits/μl, Seikagaku Kogyo) treatment was done in 0.1 m sodium acetate, pH 5.2, 0.5 mm 4-amidophenylmethylsulfonyl fluoride at 37 °C for 12 h. Native chitinase was isolated from the supernatant of the saturated cultures of S. cerevisiae grown in YEPD as described by Kuranda and Robbins (17Kuranda M.J. Robbins P.W. J. Biol. Chem. 1991; 266: 19758-19767Abstract Full Text PDF PubMed Google Scholar). The supernatant of the 10-ml culture was mixed with 30 mg of purified chitin (Sigma) to allow binding of chitinase. Chitin was then pelleted by centrifugation and washed three times with TBS (50 mm Tris, pH 7.5, 150 mm NaCl). The washed pellet was suspended in 100 μl of SDS sample buffer, heated to 100 °C for 10 min, and analyzed by SDS-polyacrylamide gel electrophoresis on 6% resolving gel. The gel was stained with Coomassie Brilliant Blue. H17–6C (vig9–1) was transformed with a yeast genomic library constructed on pRS314 (ARSH4 CEN6 TRP1; a kind gift of A. Yamamoto) by the lithium acetate method (18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Trp+ transformants were selected and maintained on SD medium and tested for complementation of the geneticin-sensitive phenotype. Plasmids were isolated from the candidate transformants and propagated in E. coli. All of the unique plasmids were retransformed into H17–6C to verify the ability to complement the glycosylation defect. Appropriate fragments were subcloned on plasmid pBluescript II SK+ (Stratagene), and deletions were constructed by a double-stranded nested deletion kit (Pharmacia). These clones were sequenced by using the Sequenase modification of the dideoxy chain termination method (19Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar) with dye primers and Applied Biosystems Inc. DNA sequencer model 373A. Sequences were analyzed with computer programs (Genetyx CD, Software Development Co., Tokyo). The disruption plasmid pSV924 was constructed by cloning the HpaI/SalI fragment containing the LEU2 gene in theHpaI/SalI site of the VIG9 gene. A diploid yeast, W303, was transformed with the Δvig9::LEU2 fragment excised from pSV924 byHindIII and SacI. Leu+ transformants were selected, and disruption of one copy of VIG9 was confirmed by Southern blotting. Spores were developed and dissected to examine essentiality of the VIG9 gene. Yeast cells were collected, washed in cold washing solution (1.4 m sorbitol, 10 mm sodium azide), and converted to spheroplasts. The spheroplasts were pelleted and resuspended in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 0.001 volume of protease inhibitor mixture (1 mg/ml each of leupeptin, chymostatin, pepstatin, aprotinin, and antipain)) and lysed osmotically. The lysate was immediately centrifuged at 750 × g for 3 min to remove debris and further centrifuged at 100,000 × gfor 1 h to produce a supernatant. The protein concentration was determined according to Bradford (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). An appropriate amount of the above cell extract was incubated in a 50-μl reaction mixture containing 40 mm HEPES, pH 7.6, 8 mm MgCl2, 0.1 mm mannose 1-phosphate, 0.2 mm GTP, 50 mm GDP, 50 mm GMP, and 0.1 mCi/ml [α-32P]GTP (specific activity 3,000 Ci/mmol, ICN) at 25 °C. Samples were withdrawn, mixed with equal amounts of stop solution (100 mm GTP, 100 mm EDTA), and frozen in dry ice-ethanol to stop the reaction. Amounts of [32P]GDP-Man formed in samples were determined with an Image Analyzer (Fuji, BAS2000) after separating the GDP-Man by chromatography on a polyethyleneimine cellulose thin layer (Merck). The amounts of radioactive materials are expressed as photostimulated luminescence, calculated by the Image Analyzer. The DNA encoding Vig9 protein was prepared by polymerase chain reaction using primer oligonucleotides 5′-CGGGATCCATGAAAGGTTTAATTTTAGTCGG and 5′-CGCTCGAGGGCGCAGAACAGATCATCA and inserted in pGEX-4T-3 (Pharmacia) after cleavage with BamHI and XhoI to construct pSV930, which has an inducible gene encoding GST-Vig9 fusion protein.E. coli BL21/pT-groE (12Yasukawa T. Kanei-Ishii C. Maekawa T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) was transformed with pSV930 or pGEX-4T-3, and the transformants were grown in 100 ml of Luria broth (1% Bacto-trypton (Difco), 0.5% Bacto yeast extract (Difco), and 0.5% NaCl) until the A 600 nm reached 0.5. Isopropyl-1-thio-β-d-galactopyranoside (0.05 mm) was added, and incubation was continued at 26.5 °C for 2 h and 4 °C for 1 h before harvest. Cell lysates were prepared by sonication, and GST-Vig9 protein or GST was adsorbed to 10 μl of glutathione-Sepharose 4B beads (Pharmacia) at 4 °C for 1 h. After five washes with 1 ml of TBS containing 1% Triton X-100, the adsorbed proteins were eluted with 0.5 ml of 50 mm Tris-HCl, pH 8.0, containing 10 mmglutathione. GDP-Man pyrophosphorylase assay was done in 50 μl of reaction mix containing 0.05 μg of GST-Vig9 protein or GST at 25 °C for 10 min. Yeast cells that were grown to A 600 nm = 1.0 in YEPD medium were transferred to YEP4G (containing 4% galactose instead of glucose in YEPD) medium containing 1 μg/ml [14C]mannose and incubated for 2 h (21Byrd J.C. Tarentino A.L. Maley F. Atkinson P.H. Trimble R.B. J. Biol. Chem. 1982; 257: 14657-14666Abstract Full Text PDF PubMed Google Scholar). Cells were then converted to spheroplasts and lyzed by suspending in lysis buffer (0.2 m sorbitol, 0.1 m NaCl, 5 mm MgCl2, 20 mm HEPES, pH 7.4, 100 μg/ml phenylmethylsulfonyl fluoride) and incubated for 30 min on ice. After centrifugation at 100,000 × g for 1 h, the supernatant was analyzed by silica thin layer chromatography (n-butyl alcohol:acetic acid:water, 4:1:2). The amount of intracellular GDP-Man was determined as the radioactivity of the spot of GDP-Man by Image Analyzer. We obtained two independent vig9 mutants among 331 spontaneous vanadate-resistant isolates. H17 (vig9–1) and J12 (vig9–2) were able to grow on YEPD medium containing up to 5 mm vanadate and on SD medium containing up to 4 mm vanadate. Both mutants grow well at either 25 or 37 °C as the parent yeast. Sensitivity of the mutants to 5-fluorouracil, hygromycin B, and cycloheximide was not altered from the parent strain. However, vig9 mutants showed hypersensitivity to geneticin (G418). They could not grow on YEPD plates containing geneticin at 50 μg/ml, but the parent yeast grew well in the presence of geneticin at 100 μg/ml. The degree of glycosylation was determined by analyzing with secretory invertase for N-glycosylation and extracellular chitinase for O-glycosylation. As shown in Fig.1 A, invertase of the vig9–1mutant migrated faster than that of the wild type in native gel electrophoresis. The difference of mobility is due to the degree of glycosylation because the invertase polypeptides showed the same mobility in SDS-polyacrylamide gel electrophoresis after removal of saccharides by endoglycosidase H treatment (Fig. 1 B).N-Glycosylation of the vig9–1 mutant was moderately defective as the rate of migration of invertase was between those of the wild type and the mnn9 mutant. EachN-linked saccharide in the mnn9 mutant is only 5 mannose residues larger than the core oligosaccharide (22Tsai P.-K. Frevert J. Ballou C.E. J. Biol. Chem. 1984; 259: 3805-3811Abstract Full Text PDF PubMed Google Scholar).O-Glycosylation was also affected in the vig9mutants (Fig. 1 B). Vanadate-resistant, geneticin-hypersensitive, and glycosylation-defective phenotypes were all recessive and cosegregated during three cycles of back-crossing. To clone the wild type VIG9 gene, we used geneticin hypersensitivity of the vig9 mutant as a selective marker. We transformed H17–6C with a genomic library of the wild type S. cerevisiae constructed on pRS314 and screened for Trp+transformants, which can grow on a YEPD plate containing geneticin at 100 μg/ml. Twenty-seven candidate transformants were tested further for vanadate sensitivity and N-glycosylation of invertase. Plasmid DNAs from four transformants that showed the wild type level of vanadate sensitivity and glycosylation of invertase were recovered inE. coli, and the restriction maps were constructed. These plasmids contained three kinds of overlapping inserts. Deletion and subcloning analysis of the fragment in pSV903 indicated that a 2.5-kilobase HindIII-Sau3A1/BamHI fragment was responsible for complementation. The 2.5-kilobase minimal fragment was cloned in a integration vector (pRS305) to make pSV921. This plasmid was integrated in the chromosome of a vig9–1/vig9–1 diploid yeast (H17W) by homologous recombination. Tetrad analysis of 26 asci of the transformant indicated that pSV921 was integrated at the vig9–1 locus. Thus, the insert contains the authentic VIG9 gene. We determined the nucleotide sequence of the 2498-base pair insert of pSV914 and found an open reading frame (ORF) of 1083 nucleotides which encodes a polypeptide of 361 amino acids. After submission of our manuscript, the complete nucleotide sequence of the S. cerevisiae genome was determined, and this ORF had a systematic name of YDL055C on the chromosome IV. The predicted molecular mass is 39,565 Da, and the pI is 5.93. There are three potential TATA sequences of the promoter elements at nucleotide −258 to −251 (TATATATA), −70 to −67 (TATA), and −14 to −8 (TATAAA) in the 5′ upstream of the ORF. If the last candidate is selected, the putative initiation methionine (nucleotides 1–3) is too close to it, and an alternate methionine at nucleotides 97–99 will be used in this case. However, this possibility is unlikely because the deduced NH2-terminal amino acid sequence is highly conserved with the NH2-terminal sequences of bacterial proteins of similar function as described below. Consensus poly(A)-polymerase recognition sequences (AATAAA) are found at nucleotides 1238–1243, 1256–1261, and 1308–1313 in the 3′ downstream of the ORF. No consensus splicing motif (5′-GTAPyGT … TACTAAC … PyAG-3′) was found in the insert of pSV914. This ORF should represent the VIG9 gene. To examine whetherVIG9 is essential for yeast cell growth, we constructed a diploid yeast in which one copy of VIG9 was disrupted. Disruption was achieved by replacing a HpaI-SalI fragment of one copy of VIG9 with LEU2 in W303 (Fig. 2 A). Disruption was confirmed by Southern blotting (Fig. 2 B). After sporulation, 31 asci were dissected on YEPD at 30 °C. Only two spores among each tetrad outgrew, and all viable colonies were auxotrophic for leucine (Fig.2 C). Another gene disruption with TRP1 also gave similar results. Thus, the VIG9 gene is essential for yeast cell growth. Fig. 3 shows the hydropathy profile of the predicted Vig9 protein. Vig9 is a hydrophilic protein without any hydrophobic stretch long enough to function as a secretory signal sequence or transmembrane domain. This suggests that Vig9 protein is a soluble cytoplasmic protein. Comparison of the predicted Vig9 protein sequence with a data base using the FASTA program (23Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar) indicated the sequence was a novel one. However, significant homology between Vig9 protein and several bacterial proteins was found (Fig. 4). Those bacterial proteins have a common characteristic. RfbF protein of Salmonella typhimurium, which is 32.7% identical in 251 amino acids to Vig9 protein, catalyzes the synthesis of CDP-glucose from glucose 1-phosphate and CTP in the biosynthetic pathway of O-antigen (24Jiang X.M. Neal Santiago B.F. Lee S.J. Romana L.K. Reeves P.R. Mol. Microbiol. 1991; 5: 695-713Crossref PubMed Scopus (276) Google Scholar). RfbF protein of Yersinia pseudotuberculosis is 31.1% identical in 257 amino acids to Vig9 protein (25Kessler A.J. Haase A. Reeves P.R. J. Bacteriol. 1993; 175: 1412-1422Crossref PubMed Google Scholar). RfbA protein ofYersinia enterocolitica is 27.6% identical in 254 amino acids and catalyzes the synthesis of dTDP-l-rhamnose. 3L. Zhang, A. Al-Hendy, P. Toivanen, and M. Skuruik, EMBL accession number S28577. StrD protein of Streptomyces griseus, which is 23.3% identical in 339 amino acids, catalyzes the synthesis of dTDP-streptose (26Distler J. Ebert A. Mansouri K. Pissowotzki K. Stockmann M. Piepersberg W. Nucleic Acids Res. 1987; 15: 8041-8056Crossref PubMed Scopus (124) Google Scholar). So, all of these enzymes catalyze the synthesis of (deoxy)NDP-sugar from sugar phosphate and (deoxy)NTP. Homology with these enzymes having this common characteristic suggests that Vig9 protein may function as GDP-Man pyrophosphorylase, which catalyzes the formation of GDP-Man from mannose 1-phosphate and GTP. Cell extracts were prepared from yeast cells and assayed for GDP-Man pyrophosphorylase activity. Fig. 5 shows that the amount of radioactive GDP-Man formed from [α-32P]GTP and mannose 1-phosphate was linearly dependent on the amount of protein in the reaction mixture. The amount of the labeled GDP-Man increased in a time-dependent manner (Fig. 6). Fig. 7 shows the effect of adding a 10-fold molar excess (2 mm) of cold NTPs on the formation of labeled GDP-Man. The authentic substrate, GTP, competitively reduced the amount of radioactive GDP-Man to 18% of that of the control reaction. The others also affected the formation of GDP-Man: ATP to 51%, UTP to 67%, and CTP to 87%. Although ATP showed relatively severe interference, we have not examined whether ADP-Man was formed or not. If the cell extract was omitted, the radioactivity in the region of the thin layer film corresponding to the spot of GDP-Man was 2.8% of the control reaction (the background count). When mannose 1-phosphate was omitted, the amount of labeled GDP-Man formed was 7.1% of the control reaction, which may indicate that only a small amount of mannose 1-phosphate is present in the cell extract. Stimulation of the reaction by the addition of pyrophosphatase to remove pyrophosphate, the other reaction product, was not significant in our assay condition (data not shown).Figure 6Activity of GDP-Man pyrophosphorylase in wild type, vig9–1 mutant, and their transformants. The enzyme activities were determined as described under "Materials and Methods" by the incorporation of [32P]GDP into GDP-Man. The protein concentration was 37 μg, and the reaction was performed at 25 °C. Samples were withdrawn from the reaction mixture at time intervals, and GDP-Man was separated by chromatography on polyethyleneimine-cellulose thin layer. Cell extracts from TM10 (wild type, ○), H17–6C (vig9, □), H17–6C/pSV914 (vig9 carrying single copy VIG9, ▪) and TM10/pSV923 (wild type carrying multicopy VIG9, •) were used as enzymes sources. A mixture of extracts from TM10 and H17–6C was also examined (▵). The amount of [32P]GDP-Man formed was expressed as relative values to that obtained with the cell extract of TM10/pSV923 at 10 min (9.1 × 10−16mol).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Competitive effect of nucleoside triphosphates. A 10-fold excess of nucleoside triphosphate (2 mm) over GTP (0.2 mm) was added to the standard reaction to examine its competition with the authentic substrate. [32P]GDP-Man formed is shown as values relative to those in the standard reaction using the lysate of TM10 (2.3 × 10−16 mol of [32P]GDP-Man was formed with 37 μg of protein after incubation at 25 °C for 10 min).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The extract of the vig9–1 mutant cells had a practically negligible activity to form GDP-Man (Fig. 6, H17–6C). This result suggests that the vig9 mutation affects the GDP-Man pyrophosphorylase activity. A mixture of equal amounts of extracts from wild type and vig9–1 cells supported the formation of the same amount of GDP-Man as did the wild type extract (Fig. 6, TM10 + H17–6C). This result indicates that the vig9 mutation did not function to produce inhibitory products or catalyze a reaction that reduces the amount of GDP-Man; this is in agreement with the recessive nature of vig9 mutations. Upon introduction of a single-copyVIG9 plasmid, the vig9 mutant cells recovered the GDP-Man pyrophosphorylase activity to the wild type level (Fig. 6, H17–6C/pSV914). Furthermore, the extract of wild type cells carrying a multicopy VIG9 plasmid showed a 3–4-fold increased activity to form GDP-Man (Fig. 6, TM10/pSV923). These results further support that VIG9 is the structural gene for GDP-Man pyrophosphorylase in accordance with the prediction from the homology between VIG9 protein and the bacterial enzymes. To demonstrate that the Vig9 protein catalyzes the formation of GDP-Man, we constructed a plasmid that produces a GST-Vig9 fusion protein as described under "Materials and Methods." The affinity-purified GST-Vig9 protein had activity to synthesize [32P]GDP-Man from [α-32P]GTP and mannose 1-phosphate, whereas the fusion partner GST did not (Fig.8). We further examined the effect of the reduced activity of GDP-Man pyrophosphorylase in the vig9 mutant. Cells of the wild type and vig9–1 mutant were grown in the presence of [14C]mannose, and the amount of GDP-Man in the cell extracts was determined. Five independent experiments demonstrated that the amount of GDP-Man in vig9–1 mutant cells was 70.6% of that of the wild type cells. This result indicates that the substrate of mannosylation is a limiting factor of glycosylation and the direct cause of the glycosylation defect in the vig9–1mutants. We have isolated a new glycosylation-defective mutation,vig9, among vanadate-resistant mutants. The biochemical rationale of this enrichment is not clear (8Ballou L. Hitzeman R.A. Lewis M.S. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3209-3212Crossref PubMed Scopus (114) Google Scholar). Mutants of a wide variety of genes including MNN8, MNN9,MNN10, ALG4/SEC53, OCH1,ANP1, VAN1, and VRG4/VAN2 showed vanadate resistance (8Ballou L. Hitzeman R.A. Lewis M.S. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3209-3212Crossref PubMed Scopus (114) Google Scholar, 27Chapman R.E. Munro S. EMBO J. 1994; 13: 4896-4907Crossref PubMed Scopus (81) Google Scholar, 28Kanik-Ennulat C. Montalvo E. Neff N. Genetics. 1995; 140: 933-943Crossref PubMed Google Scholar, 29Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).2 The ALG4/SEC53encodes phosphomannomutase (30Kepes F. Schekman R. J. Biol. Chem. 1988; 263: 9155-9161Abst
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