1887

Abstract

The synthesis and metabolism of yeast cell wall glucan were studied using a construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected. In contrast, cycloheximide, a known ps inhibitor, displayed an elevated ratio. This whole cell construct affords a simplified system for elucidating the synthesis and metabolic activity of the yeast cell wall and enables the discrimination between specific effectors of gs and ps.

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1994-09-01
2021-10-18
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References

  1. Beaudouin R., Lim S. T., Steide J. A., Powell M., McKoy J., Pramanik A.J., Johnson E., Wood-Moore C., Lipke P. 1993; Bleomycin affects cell wall anchorage of mannoproteins in Sac-charomyces cerevisiae.. Antimicrob Agents Chemother 37:1264–1269
    [Google Scholar]
  2. Bevan P., Douglas H. C. 1969; Genetic control of phospho-glucomutase variants in Saccbaromyces cerevisiae. J Bacteriol 98:532–535
    [Google Scholar]
  3. Cabib E., Roberts R., Bowers B. 1982; Synthesis of the yeast cell wall and its regulation. Ann Rev Biochem 51:763–793
    [Google Scholar]
  4. Cabib E., Silverman S. J., Shaw J. A., Das Gupta S., Park H.-M., Mullins J. T., Mol P. C., Bowers B. 1991; Carbohydrates as structural constituents of yeast cell wall and septum. Pure Appl Chem 63:483–489
    [Google Scholar]
  5. Capobianco J.O., Zakula D., Coen M. L., Goldman R. C. 1993; iKnti-Candida activity of cispentacin: the active transport by amino acid permeases and possible mechanisms of action. Biochem Biophy Res Commun 190:1037–1044
    [Google Scholar]
  6. Chambers R. S., Broughton M. J., Cannon R. D., Carne A., Emerson G. W., Sullivan P. A. 1993; An exo-beta-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene. J Gen Microbiol 139:325–334
    [Google Scholar]
  7. Elorza M.V., Sentandreu R. 1969; Effect of cycloheximide in yeast cell wall synthesis. Biochem Biophys Res Commun 36:741–747
    [Google Scholar]
  8. Farkas I., Hardy T. A., Goebl M. G., Roach P. J. 1991; Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled. J Biol Chem 266:15602–15607
    [Google Scholar]
  9. Fleet G.H., Manners D. J. 1976; Isolation and composition of an alkali-soluble glucan from the cell walls of Saccharomyces cerevisiae. J Gen Microbiol 94:180–192
    [Google Scholar]
  10. Frost D., Brandt K., Capobianco J., Goldman R. 1992; A microsomal and permeabilized whole cell assay of (1,3)-ß-D-glucan synthase from Candida albicans. Abstract F46.. Annu Meet Am Soc Microbiol 92:506
    [Google Scholar]
  11. Gancedo J.M. 1992; Carbon catabolite repression in yeast. Eur J Biochem 206:297–313
    [Google Scholar]
  12. Gooday G. W., Zhu W. Y., O'Donnell R. W. 1992; What are the roles of chitinases in the growing fungus?. FEMS Microbiol Eett 100:387–392
    [Google Scholar]
  13. Gopal P., Sullivan P. A., Shepherd M. G. 1984; Isolation and structure of glucan from regenerating spheroplasts of Candida albicans.. J Gen Microbiol 130:1217–1225
    [Google Scholar]
  14. Hartland R. P., Emerson G. W., Sullivan P. A. 1991; A secreted ß-glucan-branching enzyme from Candida albicans.. Proc R Soc Eond B 246:155–160
    [Google Scholar]
  15. Hector R.F. 1993; Compounds active against cell walls of medicallv important fungi. Clin Microbiol Rev 6:1–20
    [Google Scholar]
  16. Hien N.H., Fleet G. H. 1983; Separation and characterization of six 1,3-ß-glucanases from Saccharomyces cerevisiae. J Bacteriol 156:1204–1213
    [Google Scholar]
  17. Ito H., Fukuda Y., Murata K., Kimura A. 1983; T ransformation of intact veast cells treated with alkali cations. J Bacteriol 153:163–168
    [Google Scholar]
  18. Klebl F., Tanner W. 1989; Molecular cloning of a cell wall exo-A-1,3-glucanase from Saccharomyces cerevisiae. J Bacteriol 171:6259–6264
    [Google Scholar]
  19. Kurranda M.J., Robbins P. W. 1991; Chitinase is required for cell separation during growth of Saccharomyces cerevisiae.. J Biol Chem 266:19758–19767
    [Google Scholar]
  20. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Manners D. J., Masson A. J., Patterson J. C. 1973a; The structure of a ß-(1-3)-D-glucan from yeast cell walls. Biochem J 135:19–30
    [Google Scholar]
  22. Manners D. J., Masson A. J., Patterson J. C., Bjorndal H., Lindberg B. 1973b; The structure of a ß-(1-6)-D-glucan from yeast cell walls. Biochem J 135:31–36
    [Google Scholar]
  23. Mrsa V., Klebl F., Tanner W. 1993; Purification and characterization of the Saccharomyces cerevisiae Bgl2 gene product, a cell wall endo-ß-1,3-glucanase.. J Bacteriol 175:2102–2106
    [Google Scholar]
  24. Nehlin J. O., Carlberg M., Ronne H. 1991; Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J 10:3373–3377
    [Google Scholar]
  25. Oh D., Hopper J. E. 1990; Transcription of a yeast phospho-glucomutase isozyme gene is galactose inducible and glucose repressible. Mol Cell Biol 10:1415–1422
    [Google Scholar]
  26. Peberdy J.F. 1990; Fungal cell walls -a review. In Biochemistry of Cell Walls and Membranes in Fungi pp. 5–30 Edited by Kuhn P. J., Trinci A. P. J. , Jung M. J. , Goosey M. W. , Copping L. G. . Berlin: Springer-Verlag;
    [Google Scholar]
  27. Pitson S. M., Seviour R. J., McDougall B. M. 1993; Non-cellulolytic fungal ß-glucanases: their physiology and regulation. Ensymol Microbiol Technol 15:178–192
    [Google Scholar]
  28. Rose M., Albig W., Entian K. D. 1991; Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII.. Eur J Biochem 199:511–518
    [Google Scholar]
  29. Ruiz-Herrera J. 1992 Fungal Cell Wall: Structure Synthesis and Assembly. Boca Raton: CRC Press;
    [Google Scholar]
  30. San Segundo P., Correa J., Vasquez De Aldana C.R., Del Rey F. 1993; SSG1, a gene encoding a sporulation-specifk 1,3-ß-glucanase in Saccharomyces cerevisiae. J Bacteriol 175:3823–3837
    [Google Scholar]
  31. Shepherd M. G., Surarit R., Gopal P. K., Sullivan P. A. 1984; Cell wall metabolism of C. albicans. ß(1-3) and ß(1-6) glucan synthesis. In Microbial Cell Wall Synthesis and Autolysis pp. 73–83 Edited by Nombela C. New York: Elsevier;
    [Google Scholar]
  32. Sietsma J.H., Wessels J. G. H. 1979; Evidence for covalent linkages between chitin and ß-glucan in a fungal cell wall. J Gen Microbiol 114:99–108
    [Google Scholar]
  33. Sietsma J.H., Wessels J. G. H. 1981; Solubility of (1-3)-ß-D-(1-6)-ß-D-glucan in fungal cell walls: importance of presumed linkage between glucan and chitin. J Gen Microbiol 125:209–212
    [Google Scholar]
  34. Surarit R., Shepherd M. G. 1987; The effects of azole and polyene antifungals on the plasma membrane enzymes of Candida albicans.. J Med Vet Mycol 25:403–413
    [Google Scholar]
  35. Surarit R., Gopal P. K., Shepherd M. G. 1988; Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans. J Gen Microbiol 134:1723–1730
    [Google Scholar]
  36. Tkacz J.S. 1984; In vivo synthesis of ß-1,6-glucan in Saccharomyces cerevisiae.. In Microbial Cell Wall Synthesis and Autolysis pp. 287–295 Edited by Nombela C. New York: Elsevier;
    [Google Scholar]
  37. Valentin E., Herrero E., Javier Paster F. I., Sentandreu R. 1984; Solubilization and analysis of mannoprotein molecules from the cell wall of Saccharomyces cerevisiae. J Gen Microbiol 130:1419–1428
    [Google Scholar]
  38. Wessels J.G.H., Mol P. C., Sietsma J. H., Vermeulen C. A. 1990; Wall structure, wall growth and fungal morphogenesis. In Biochemistry of Cell Walls and Membranes in Fungi pp. 81–95 Edited by Kuhn P. J., Trinci A. P. J. , Jung M. J. , Goosey M. W. , Copping L. G. . Berlin: Springer-Verlag;
    [Google Scholar]
  39. Winchester B., Fleet G. W. J. 1992; Amino-sugar glycosidase inhibitors: versatile tools for glycobiologists. Glycobiology 2:199–210
    [Google Scholar]
  40. Yu L. P., Goldman R., Sullivan P., Walker G. F., Fesik S. W. 1993; Heteronuclear nmr studies of C-13-labelled cell wall ß-glucan oligosaccharides. J Biomolecular NMR 3:429–441
    [Google Scholar]
  41. Yun S. J., Hiraoka Y., Nishizawa M., Takio K., Titani K., Nogi Y., Fukasawa T. 1991; Purification and characterization of the yeast negative regulatory protein GAL80. J Biol Chem 266:693–697
    [Google Scholar]
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