1887

Abstract

Inducible overexpression of the gene under the control of the promoter increased Chs3p (chitin synthase 3) activity in several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP--acetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein–protein interaction.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-2-385
2000-02-01
2020-01-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/2/1460385a.html?itemId=/content/journal/micro/10.1099/00221287-146-2-385&mimeType=html&fmt=ahah

References

  1. Bulawa C. E.. 1992; CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis. Mol Cell Biol12:1764–1776
    [Google Scholar]
  2. Bulawa C. E.. 1993; Genetics and molecular biology of chitin synthesis in fungi. Annu Rev Microbiol47:505–534[CrossRef]
    [Google Scholar]
  3. Bulawa C. E., Slater M., Cabib E., Au-Young J., Sburlati A., Adair W. L. Jr, Robbins P. W.. 1986; The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo. Cell46:213–225[CrossRef]
    [Google Scholar]
  4. Cabib E., Sburlati A., Bowers B., Silverman S. J.. 1989; Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae. J Cell Biol108:1655–1672
    [Google Scholar]
  5. Cabib E., Silverman S. J., Shaw J. A.. 1992; Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae. J Gen Microbiol138:97–102[CrossRef]
    [Google Scholar]
  6. Cabib E., Shaw J. A., Mol P. C., Bowers B., Choi W.-J.. 1996; In The Mycota, vol. III, Biochemistry and Molecular Biology pp.243–267Edited by Brambl R., Marzluf G. A.. Berlin, Heidelberg & New York: Springer;
    [Google Scholar]
  7. Choi W.-J., Sburlati A., Cabib E.. 1994; Chitin synthase 3 from yeast has zymogenic properties that depend on both the CAL1 and the CAL3 genes. Proc Natl Acad Sci USA91:4727–4730[CrossRef]
    [Google Scholar]
  8. Chuang J. S., Schekman R. W.. 1996; Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p. J Cell Biol135:597–610[CrossRef]
    [Google Scholar]
  9. Cos T., Ford R., Trilla A., Duran A., Cabib E., Roncero C.. 1998; Molecular analysis of Chs3p participation in chitin synthase III activity. Eur J Biochem256:419–426[CrossRef]
    [Google Scholar]
  10. DeMarini D. J., Adams A. E. M., Fares H., Virgilio C. D., Valle G., Chuang J. S., Pringle J. R.. 1997; A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall. J Cell Biol139:75–93[CrossRef]
    [Google Scholar]
  11. Feilotter H. E., Hannon G. J., Ruddell C. J., Beach D.. 1994; Construction of an improved host strain for two hybrid screening. Nucleic Acids Res22:1502–1503[CrossRef]
    [Google Scholar]
  12. Holthuis J. C. M., Nichols B. J., Pelham H. R. B.. 1998; The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast. Mol Biol Cell9:3383–3397[CrossRef]
    [Google Scholar]
  13. Ito H., Fukuda Y., Murata K., Kimura A.. 1983; Transformation of intact yeast cells treated with alkali cations. J Bacteriol153:163–168
    [Google Scholar]
  14. Kawamoto S., Nomura M., Ohno T.. 1992; Cloning and characterization of SKT5, a Saccharomyces cerevisiae gene that affects protoplast regulation and resistance to killer toxin of Kluyveromyces lactis. J Ferment Bioeng74:199–208
    [Google Scholar]
  15. Kollar R., Petrakova E., Ashwell G., Robbins P. W., Cabib E.. 1995; Architecture of the yeast cell wall. The linkage between chitin and beta (1→3)-glucan. J Biol Chem270:1170–1178[CrossRef]
    [Google Scholar]
  16. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  17. Nagahashi S., Sudoh M., Ono N..7 other authors 1995; Characterization of chitin synthase 2 of Saccharomyces cerevisiae: implication of two highly conserved domains as possible catalytic sites. J Biol Chem270:13961–13967[CrossRef]
    [Google Scholar]
  18. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. pp.18.60–18.75 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Santos B., Snyder M.. 1997; Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p. J Cell Biol136:95–110[CrossRef]
    [Google Scholar]
  20. Santos B., Duran A., Valdivieso M. H.. 1997; CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae. Mol Cell Biol17:2485–2496
    [Google Scholar]
  21. Sburlati A., Cabib E.. 1986; Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae. J Biol Chem261:15147–15152
    [Google Scholar]
  22. Shaw J. A., Mol P. C., Bowers B., Silverman S. J., Valdivieso M. H., Duran A., Cabib E.. 1991; The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol114:111–123[CrossRef]
    [Google Scholar]
  23. Silverman S. J.. 1989; Similar and different domains of chitin synthases 1 and 2 of S. cerevisiae: two isozymes with distinct functions. Yeast5:459–467[CrossRef]
    [Google Scholar]
  24. Silverman S. J., Sburlati A., Slater M. L., Cabib E.. 1988; Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae. Proc Natl Acad Sci USA85:4735–4739[CrossRef]
    [Google Scholar]
  25. Sudoh M., Tatsuno K., Ono N., Chibana H., Yamada-Okabe H., Arisawa M.. 1999; The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis. Microbiology145:1613–1622[CrossRef]
    [Google Scholar]
  26. Takita M., Castilho-Valvicius B.. 1993; Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin. Yeast9:589–598[CrossRef]
    [Google Scholar]
  27. Trilla J. A., Cos T., Duran A., Roncero C.. 1997; Characterization of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4. Yeast13:795–807[CrossRef]
    [Google Scholar]
  28. Valdivieso M. H., Mol P. C., Shaw J. A., Cabib E., Duran A.. 1991; CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae. J Cell Biol114:101–109[CrossRef]
    [Google Scholar]
  29. Yabe T., Yamada-Okabe T., Nakajima T., Sudoh M., Arisawa M., Yamada-Okabe H.. 1998; Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae; identification of additional amino acid residues involved in the catalysis. Eur J Biochem258:941–947[CrossRef]
    [Google Scholar]
  30. Ziman M., Chuang J. S., Schekman R. W.. 1996; Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway. Mol Biol Cell7:1909–1919[CrossRef]
    [Google Scholar]
  31. Ziman M., Chung J. S., Tsung M., Hashimoto S., Schekman R.. 1998; Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae. Mol Biol Cell9:1565–1576[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-2-385
Loading
/content/journal/micro/10.1099/00221287-146-2-385
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error