DOI QR코드

DOI QR Code

Saccharomyces cerevisiae의 KGD1 유전자 결손이 세포벽 생합성에 미치는 영향

Effect of KGD1 Deletion on Cell Wall Biogenesis in Saccharomyces cerevisiae

  • 김성우 (충남대학교 생명시스템과학대학 미생물.분자생명과학과) ;
  • 안기웅 (충남대학교 생명시스템과학대학 미생물.분자생명과학과) ;
  • 박윤희 (충남대학교 생명시스템과학대학 미생물.분자생명과학과) ;
  • 박희문 (충남대학교 생명시스템과학대학 미생물.분자생명과학과)
  • Kim, Sung-Woo (Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University) ;
  • Ahn, Ki-Woong (Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University) ;
  • Park, Yun-Hee (Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University) ;
  • Park, Hee-Moon (Department of Microbiology & Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University)
  • 투고 : 2010.06.02
  • 심사 : 2010.06.17
  • 발행 : 2010.06.30

초록

KGD1 유전자는 비허용온도에서 세포벽에 결함을 보이는 Saccharomyces cerevisiae LP0353 균주의 베타-1,3-글루칸 합성 효소의 활성을 회복시키는 유전자로 분리되었다. $\alpha$-ketoglutarate dehydrogenase를 암호화하는 KGD1 유전자의 효모의 세포벽 합성과 연관된 기능을 분석하기 위하여 유전자 파괴를 시도하였다. KGD1돌연변이는 생장속도가 감소하고, 키틴 합성 효소들의 활성이 증가하였으며, 세포벽 구성 당류의 함량에 변화를 보였다. 또한 Calcofluor white과 Nikkomycin Z 등과 같은 세포벽 합성 저해물질에 대해 감수성 변화를 나타냈다. 이러한 결과들은 KGD1이 효모의 세포벽 특히 베타-1,6-글루칸과 키틴의 생합성에 영향을 주고 있음을 시사한다.

KGD1 gene was cloned by functional complementation of defects in $\beta$-1,3-glucan synthase activity of the previously isolated Saccharomyces cerevisiae mutant LP0353, which displays a number of cell wall defects at restrictive temperature. We performed the gene disruption experiment to characterize the function of KGD1 gene, which encodes $\beta$-ketoglutarate dehydrogenase, in cell wall biosynthesis. The disruption of KGD1 showed the decreased growth rate, the increase of chitin synthases activity, alterations in cell wall composition, and increase of susceptibility to cell wall inhibitors such as Calcofluor white and Nikkomycin Z. These results suggested that KGD1 might be involved in cell wall biogenesis, especially the biosynthesis of $\beta$-1,6-glucan and chitin in S. cerevisaie.

키워드

참고문헌

  1. 이동원, 박상원, 진은희, 정재훈, 김진미, 박희문. 1994. 베타-1,3-글루칸 합성능이 손상된 Saccharomyces cerevisiae 돌연변이주의 유전적 분석. 한국유전학회지 16:259-268
  2. 진은희, 이동원, 김진미, 박희문. 1995. 베타-1,3-글루칸 생합성에 관여하는 Saccharomyces cerevisiae 유전자의 클로닝. 한국균학회지 23:129-138.
  3. Ahn, K.-W., Kim, S. W., Kang, H.-G., Kim, K.-H., Park, Y.-H., Choi, W.-J. and Park, H.-M. 2010. Deletion of GBG1/AYR1 Alters Cell Wall Biogenesis in Saccharomyces cerevisiae. Mycobiol 38: In press.
  4. Arikawa, Y., Kuroyanagi, T., Shimosaka, M., Muratsubaki, H., Enomoto, K., Kodaira, R. and Okazaki, M. 1999. Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae. J Biosci Bioeng 87:28-36. https://doi.org/10.1016/S1389-1723(99)80004-8
  5. Athenstaedt, K. and Daum, G. 2000. 1-Acyldihydroxyacetonephosphate reductase (Ayr1p) of the yeast Saccharomyces cerevisiae encoded by the open reading frame YIL124w is a major component of lipid particles. J Biol Chem 275:235-240. https://doi.org/10.1074/jbc.275.1.235
  6. Burke, D., Dawson, D. and Stearns, T. 2000. Methods in Yeast Genetics : A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press.
  7. Cabib, E., Bowers, B., Sburlati, A. and Silverman, S. J. 1988. Fungal cell wall synthesis: the construction of a biological structure. Microbiol Sci 5:370-375.
  8. Choi, W. J. and Cabib, E. 1994. The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Anal Biochem 219:368-372. https://doi.org/10.1006/abio.1994.1278
  9. de Groot, P. W., Ruiz, C., Vazquez de Aldana, C. R., Duenas, E., Cid, V. J., Del Rey, F., Rodriquez-Pena, J. M., Perez, P., Andel, A., Caubin, J., Arroyo, J., Garcia, J. C., Gil, C., Molina, M., Garcia, L. J., Nombela, C. and Klis, F. M. 2001. A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics 2:124-142. https://doi.org/10.1002/cfg.85
  10. el-Sherbeini, M. and Clemas, J. A. 1995. Nikkomycin Z supersensitivity of an echinocandin-resistant mutant of Saccharomyces cerevisiae. Antimicrob Agents Chemother 39:200-207. https://doi.org/10.1128/AAC.39.1.200
  11. Hohmann, S., Mager, W. H., Gasch, A. P., Trott, A., Morano, K. A., Tamas, M. J., Wadskog, I., Adler, L., Toledano, M. B., Delaunay, A., Biteau, B., Spector, D., Azevedo, D., Winderickx, J., Holsbeeks, I., Lagatie, O., Giots, F., Thevelein, J. and Winde, H. d. 2003. Yeast Stress Response, Springer.
  12. Ishiguro, J., Saitou, A., Duran, A. and Ribas, J. C. 1997. cps1+,a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B. J Bacteriol 179:7653-7662. https://doi.org/10.1128/jb.179.24.7653-7662.1997
  13. Ito, H., Fukuda, Y., Murata, K. and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163-168.
  14. Kang, M. S. and Cabib, E. 1986. Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1${\to}$3)-beta-D-glucan synthase. Proc Natl Acad Sci USA 83:5808-5812. https://doi.org/10.1073/pnas.83.16.5808
  15. Kurtz, M. B., Douglas, C., Marrinan, J., Nollstadt, K., Onishi, J., Dreikorn, S., Milligan, J., Mandala, S., Thompson, J., Balkovec, J. M. and et al. 1994. Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis. Antimicrob Agents Chemother 38:2750-2757. https://doi.org/10.1128/AAC.38.12.2750
  16. Latge, J. P. 2007. The cell wall: a carbohydrate armour for the fungal cell. Mol Microbiol 66:279-290. https://doi.org/10.1111/j.1365-2958.2007.05872.x
  17. Lee, D.-W., Ahn, G.-W., Kang, H.-G. and Park, H.-M. 1999. Identification of a gene, SOO1, which complements osmo-sensitivity and defect in vitro beta-1,3-glucan synthase activity in Saccharomyces cerevisiae. Biochim Biophys Acta 1450:145-154. https://doi.org/10.1016/S0167-4889(99)00041-5
  18. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.
  19. Lussier, M., White, A. M., Sheraton, J., di Paolo, T., Treadwell, J., Southard, S. B., Horenstein, C. I., Chen-Weiner, J., Ram, A. F., Kapteyn, J. C., Roemer, T. W., Vo, D. H., Bondoc, D. C., Hall, J., Zhong, W. W., Sdicu, A. M., Davies, J., Klis, F. M., Robbins, P. W. and Bussey, H. 1997. Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147:435-450.
  20. Manners, D. J., Masson, A. J. and Patterson, J. C. 1973. The structure of a beta-(1→3)-D-glucan from yeast cell walls. Biochem J 135:19-30. https://doi.org/10.1042/bj1350019
  21. Repetto, B. and Tzagoloff, A. 1989. Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase. Mol Cell Biol 9:2695-2705. https://doi.org/10.1128/MCB.9.6.2695
  22. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: a Laboratory Manual, 2nd edition, Cold Spring Horbor Laboratory Press, New York.
  23. Smaczynska-de Rooij, I., Migdalski, A. and Rytka, J. 2004. Alpha-Ketoglutarate dehydrogenase and lipoic acid synthase are important for the functioning of peroxisomes of Saccharomyces cerevisiae. Cell Mol Biol Lett 9:271-286.
  24. Song, M. R., Lee, D. W., Park, S. W., Bae, K. S. and Park, H. M. 1992. Isolation and characterization of Saccharomyces cerevisiae mutants deficient in (1→3)-beta-D-Glucan synthase. Kor J Appl Microbiol Biotechnol 20:642-646.
  25. Spreghini, E., Davis, D. A., Subaran, R., Kim, M. and Mitchell, A. P. 2003. Roles of Candida albicans Dfg5p and Dcw1p cell surface proteins in growth and hypha formation. Eukaryot Cell 2:746-755. https://doi.org/10.1128/EC.2.4.746-755.2003
  26. Tracey, M. V. 1956. Chitin. In: Modern Methods of Plant Analysis. Eds. P. Peach and M. V. Tracey. Springer-Verlag. 264-274.
  27. Vink, E., Vossen, J. H., Ram, A. F., van den Ende, H., Brekelmans, S., de Nobel, H. and Klis, F. M. 2002. The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae. Microbiol 148:4035-4048. https://doi.org/10.1099/00221287-148-12-4035
  28. Zahedi, R. P., Sickmann, A., Boehm, A. M., Winkler, C., Zufall, N., Schonfisch, B., Guiard, B., Pfanner, N. and Meisinger, C. 2006. Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins. Mol Biol Cell 17:1436-1450. https://doi.org/10.1091/mbc.E05-08-0740