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Keywords:

  • carbon metabolism;
  • UDP-glucose;
  • cell wall;
  • reserve carbohydrates;
  • Saccharomyces cerevisiae

Abstract

  1. Top of page
  2. Abstract
  3. References

We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977–986] encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1–phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent. On glucose medium, the 40–fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth. In contrast, on galactose, the 40–fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3–5–fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1–phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5–10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10–fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall β-glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism.

Abbreviations
Glc-1P

glucose 1-phosphate

Glc-6P

glucose-6-phosphate

Gal-1P

galactose-1-phosphate

UGPase

UDP-glucose pyrophoapholylase

PPi

pyrophosphate

Enzymes
 

UDP-Glc pyrophosphorylase (EC 2.7.7.9)

 

UDP-Glc-4 epimerase (EC 5.1 2.3)

 

phosphoglucomutase (EC 5.4.2.2)

 

glucose-6-phosphate dehydrogenase (EC 1.1.1.49)

 

trehalase (EC 3.2.1.28)

References

  1. Top of page
  2. Abstract
  3. References
  • Bergmeyer, H. U. (1986) Methods in enzymatic analysis, 3rd edn, Verlag Chemie, Weinheim.
  • Boone, C., Sommer, S. S., Hensel, A. & Bussey, H. (1990) Yeast KRE genes provide evidence for a pathway of cell wall β-glucan assembly, J. Cell. Biol. 110, 18331843.
  • Bradford, M. A. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248254.
  • Cabib, E. & Kang, M. S. (1987) Fungal 1,3-β-glucan synthase, Methods Enzymol. 138, 637642.
  • Chomczynski, P. & Sacchi, N. (1987) Single step method of RNA isolation by acid guanidium isothyocyanate-phenol-chloroform extraction, Anal. Biochem. 162, 156159.
  • Douglas, C. M., Marrinan, J. A., Li, W. & Kurtz, M. B. (1994) A Saccharomyces cerevisiae mutant with echinocandine-resistant 1,3-β-D-glucan synthase, J. Bacterial. 176, 56865696.
  • Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Colorimetric method for determination of sugars and related substances, Anal. Chem. 28, 350356.
  • Entian, K. D., Zimmermann, F. K. & Scheel, I. (1977) A partial defect in carbon catabolite repression in mutants of Saccharomyces cerevisiae with reduced hexose phosphorylation, Mol. Gen. Genet. 156, 99105.
  • François, J., Eraso, P. & Gancedo, C. (1987) Changes in the concentration of cAMP, fructose-2,6–bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae, Eur. J. Biochem. 164, 369373.
  • Francois, J. & Hers, H. G. (1988) The control of glycogen metabolism in yeast. 2: A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their intercon-version in a cell-free extract, Eur. J. Biochem. 174, 561567.
  • Fukasawa, T., Sagawa, T. & Nogi, Y. (1982) Uridine diphosphate glu-cose-4-epimerase and galactose-1–phosphate uridylyltransferase from Saccharomyces cerevisiae, Methods Enzymol. 89, 584588.
  • Gancedo, J. M. & Gancedo, C. (1973) Concentration of intermediary metabolites in yeast, Biochimie (Paris) 55, 205211.
  • Guthrie, C. & Fink, G. R., ed. (1991) Guide to yeast genetics and molecular biology, Methods Enzymol. 194, Academic Press, San Diego .
  • Hartland, R. P., Vermeulen, C. A., Klis, F. M., Siestsma, J. H. & Wessels, J. G. H. (1994) The linkage of (1-3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae, Yeast 10, 15911599.
  • Herscovics, A. & Orlean, P. (1993) Glycoprotein biosynthesis in yeast, FASEB. J. 7, 540550.
  • Hill, J. E., Myers, A. M., Koerner, T. J. & Tzagaloff, A. (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites, Yeast 2, 163167.
  • Hong, Z., Mann, P., Brown, N. H., Tran, L. E., Shaw, K. J., Hare, R. S. & Didomenico, B. (1994a) Cloning and characterization of KNR4, a yeast gene involved in (1,3)-β-glucan synthesis, Mol. Cell. Biol. 14, 10171025.
  • Hong, Z., Mann, P., Shaw, K. J. & Didomenico, B. B. (1994b) Analysis of β-glucan and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography, Yeast 10, 10831092.
  • Johnston, M. (1987) A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae, Microbiol. Rev. 51, 458476.
  • Johnston, M., Andrews, S., Brinkman, R., Cooper, J., Ding, H., Dover, J., Du, Z., Favello, L., Gattung, S., Geisel, C., Kirsten, J., Kucaba, T., Hillier, L., Jier, M., Johnston, L., Langston, Y., Latreille, P., Louis, E. J., Macri, C., Mardis, E., Meneze, L., Mouser, L., Nhan, M., Rifkin, L., Riles, L., St Peter, H., Trevaskis, T., Vaughan, K., Vignati, D., Wilcox, L., Wolhdman, P., Waterston, R., Wilson, R. & Vaudin, M. (1994) Complete nucleotide sequence of Saccharomyces cerevisiae chromosome VIII, Science 265, 20772082.
  • Katsube, T., Kazuta, Y., Mori, H., Nakano, K., Tanizawa, K. & Fukui, T. (1990) UDP-Glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing, J. Biochem. 108, 321326.
  • Katsube, T., Kazuta, Y., Tanizawa, K. & Fukui, T. (1991) Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site, Biochemistry 30, 85468551.
  • Kazuta, Y., Omura, Y., Tagaya, M., Nakano, K. & Fukui, T. (1991) Identification of lysyl residues located at the substrate-binding site in UDP-Glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals, Biochemistry 30, 85418545.
  • Klis, F. (1994) Cell wall assembly in Yeast, Yeast 10, 851869.
  • Konishi, Y., Tanizawa, K., Muroya, S. & Fukui, T. (1993) Molecular cloning nucleotide sequencing and affinity labeling of bovine liver UDP-glucose pyrophosphorylase, J. Biochem. 114, 6168.
  • Laemmli, U. K. (1970) Cleavage of structural proteins during the assemble of the head of bacteriophage T4, Nature 227, 680685.
  • Lagunas, R. & Moreno, E. (1992) Inhibition of glycolysis by 2-deoxy-galactose in Saccharomyces cerevisiae. Yeast 8, 107115.
  • Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265275.
  • Montijn, R. C., van Rinsum, J., van Schagen, F. A. & Klis, F. M. (1994) Glucomannoproteins in cell wall of Saccharomyces cerevisiae contain a novel type of carbohydrate side chain, J. Biol. Chem. 269, 1933819342.
  • Panek, A. (1990) Storage carbohydrates, in The yeasts (Rose, A. H. & Harrison, J. S., eds) 2nd edn, vol. 4, pp. 655678, Academic Press, San Diego .
  • Peng, H. L. & Chang, H. Y. (1993) Cloning of a human liver UDP-glucose pyrophosphorylase cDNA by complementation of the bacterial galU mutation. FEBS Lett. 329, 153158.
  • Platt, T. (1984) Toxicity of 2–deoxygalactose to Saccharomyces cerevisiae cells constitutively synthesizing galactose-metabolizing enzymes, Mol. Cell. Biol. 4, 994996.
  • Purnelle, B., Skala, J., Van Dijek, L. & Goffeau, A. (1992) The sequence of a 12–kb fragment on the left arm of yeast chromosome XI reveals five new open reading frames, including a zinc finger protein and a homolog of the UDP-glucose pyrophosphorylase from potato, Yeast 8, 977986.
  • Ragheb, J. A. & Dottin, P. R. (1987) Structure and sequences of a UDP-glucose pyrophosphorylase gene of Dictyostelium discoideum, Nucleic Acids Res. 15, 38913908.
  • Ram, A. F. J., Wolters, A., Ten Hooper, R. & Klis, F. (1994) A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white, Yeast 10, 10191030.
  • Rhodes, N., Company, M. & Errede, B. (1990) A yeast-Escherichia coli shuttle vector containing the M13 origin of replication, Plasmid 23, 159162.
  • Robzyk, K. & Kassir, Y. (1992) A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acid Res. 20, 3790.
  • Roemer, T. & Bussey, H. (1991) Yeast β-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro, Proc. Natl Acad. Sci. USA. 88, 1129511299.
  • Roemer, T., Delaney, S. & Bussey, H. (1993) SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in β-glucan synthesis, Mol. Cell. Biol. 13, 40394048.
  • Rose, M. D., Winston, F. & Hieter, P. (1990) Methods in yeast genetics. A laboratory course manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York .
  • Rothstein, R. J. (1983) One step gene disruption in yeast, Methods Enzymol. 101, 202211.
  • Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, CSH, New York .
  • Schiestl, R. H. & Gietz, H. S. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acid as a carrier, Curr. Genet. 16, 339346.
  • Soldo, B., Lazarevic, V., Margot, P. & Karamata, D. (1993) Sequencing and analysis of the divergon comprising gtaB, the structural gene of UDP-glucose pyrophosphorylase of Bacillus subtilis 168, J. Gen. Microbiol. 139, 31853195.
  • Stagljar, I., te Heesen, S. & Aebi, M. (1994) New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus, Proc. Natl Acad. Sci. USA 91, 59775981.
  • Stratford, M. (1992) Yeast flocculation: a new perspective, Adv. Microbiol. Physiol. 33, 271.
  • te Hecsen, S., Lehle, L., Weissmann, A. & Aebi, M. (1994) Isolation of ALG5 locus encoding the UDP-glucose: dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae, Eur. J. Biochem. 224, 7179.
  • Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22, 46734680.
  • Turnquist, R. L. & Hansen, R. G. (1973) Uridine diphosphoryl glucose pyrophosphorylase, The enzyme, vol. 8, pp. 5172, Academic Press. New York & London .
  • Vandercammen, A., François, J., Torres, B. B., Maia, J. C. C. & Hers, H. G. (1990) Fructosc-2,6–bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii, J. Gen. Microbiol. 136, 137146.
  • Weissborn, A. E., Liu, Q., Rumley, M. K. & Kennedy, E. P. (1994) UTP:a-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: Isolation and DNA sequence of the galU gene and purification of the enzyme, J. Bacterial. 176, 26112618.
  • Werner-Washburne, M., Braun, E., Johnston, G. C. & Singer, R. A. (1993) Stationary phase in the yeast Saccharomyces cerevisiae, Microbiol. Rev. 57, 383401.
  • Yao, B., Marmur, J. & Sollitti, P. (1993) Construction of glucose-repressible yeast expression vectors, Gene (Amst.) 139, 223226.