Laboratory of Applied Stress MicrobiologyLaboratory of Applied Stress Microbiology

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Research Projects

1. Novel stress-tolerant mechanisms of yeast cells and their applications for breeding of industrial yeast

4) Ubiquitin system: Degradation and repair mechanisms of stress-induced abnormal proteins, regulation of the uniqutin ligase Rsp5 activity

We are also studying the ubiquitin (Ub) system under stress conditions. Rsp5 is an essential E3 ubiquitin ligase and participates in many biological events. This enzyme contains the catalytic HECT-domain that ligates Ub to the target protein and the WW domains, which recognize and interact with the target protein. We previously isolated an interesting mutant, which is hypersensitive to various stresses that induce protein misfolding. This mutant has a single amino acid change within the WW domain (A401E). It is quite interesting whether or how Rsp5 is involved in these processes. We showed that Rsp5 plays pivotal roles in both repair and degradation of stress-induced abnormal proteins. Rsp5 is suggested to regulate the expression of transcription factors that induce molecular chaperons via the nuclear export of RNA molecules. Recently, Rsp5 was found to down-regulate the cytoplasmic and plasma membrane proteins, Egd2 and Gap1, respectively, in response to high temperature or ethanol treatment through ubiquitination. In particular, we hypothesize that Rsp5 is involved in selective ubiquitination for quality control of plasma membrane proteins under stress conditions.

  1. A. Sangkaew, T. Kojornna, R. Tanahashi, H. Takagi and C. Yompakdee: A novel yeast-based screening system for potential compounds that can alleviate human α-synuclein toxicity. J. Appl. Microbiol., 132, 1409-1421 (2022).
  2. R. Tanahashi, T. Matsushita, A. Nishimura and H. Takagi: Downregulation of the broad-specificity amino acid permease Agp1 mediated by the ubiquitin ligase Rsp5 and the arrestin-like protein Bul1 in yeast. Biosci. Biotech. Biochem., 85, 1266-1274 (2021).
  3. R. Tanahashi, T. S. N.Afiah, A. Nishimura, D. Watanabe and H. Takagi*: The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source. FEMS Yeast Res., 20, foaa058, DOI: 10.1093/femsyr/foaa058.
  4. J. Yoshidaa, Y. Okawa, T. Oyama, S. Uesugi, H. Takagi, Y. Ito, and K. Kimura: Inhibition of calcineurin and glycogen synthase kinase-3β by ricinoleic acid derived from castor oil. Lipids, 55, 89-99 (2020).
  5. A. Watcharawipas, D. Watanabe and H. Takagi: Sodium acetate responses in Saccharomyces cerevisiae and the ubiquitin ligase Rsp5. Front. Microbiol., 9, 2495 (2018).
  6. D. T. Minh, H. Takagi and Y. Kimata: Categorization of endoplasmic reticulum stress as accumulation of unfolded proteins or membrane lipid aberrancy using yeast Ire1 mutants. Biosci. Biotech. Biochem., 83, 1-4 (2018).
  7. D. Watanabe, H. Sekiguchi, Y. Sugimoto, A. Nagasawa, N. Kida and H. Takagi: Importance of proteasome gene expression during model dough fermentation after freezing preservation of baker’s yeast cells. Appl. Environ. Microbiol., 84(12), e00406-18 (2018).
  8. C. T. Mai, Q. G. Le, Y. Ishiwata-Kimata, H. Takagi, K. Kohno and Y. Kimata: 4-Phenylbutyrate suppresses the unfolded protein response without restoring protein folding in Saccharomyces cerevisiae. FEMS Yeast Res., 18, foy016 (2018).
  9. A. Watcharawipas, D. Watanabe and H. Takagi: Enhanced sodium acetate tolerance in Saccharomyces cerevisiae by the Thr255Ala mutation of the ubiquitin ligase Rsp5. FEMS Yeast Res., 17, fox083 (2017).
  10. D. Watanabe, H. Murai, R. Tanahashi, K. Nakamura, T. Sasaki and H. Takagi: Cooperative and selective roles of the WW domains of the yeast Nedd4-like ubiquitin ligase Rsp5 in the recognition of the arrestin-like adaptors Bul1 and Bul2. Biochem. Biophys. Res. Commun., 463, 76-81 (2015).
  11. I. Wijayanti*, D. Watanabe*, S. Oshiro and H. Takagi: Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human α-synuclein. *These authors contributed equally to this work. J. Biochem., 157, 251-260 (2015).
  12. S. Oshiro and H. Takagi: The transcriptional activator Pog1 controls cell cycle and its phosphorylated form is downregulated by the ubiquitin ligase Dma2 in Saccharomyces cerevisiae. FEMS Yeast Res., 14, 1015-1027 (2014).
  13. T. Shiga, N. Yoshida, Y. Shimizu, E. Suzuki, T. Sasaki, D. Watanabe and H. Takagi: Quality control of plasma membrane proteins by yeast Nedd4-like ubiquitin ligase Rsp5p under environmental stress conditions. Eukaryot. Cell, 13, 1191-1199 (2014).
  14. S. Uesugi, D. Watanabe, M. Kitajima, R. Watanabe, Y. Kawamura, M. Ohnishi, H. Takagi and K. Kimura: Calcineurin inhibitors suppress the high-temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: A new method of screening for calcineurin inhibitors. FEMS Yeast Res., 14, 567-574 (2014).
  15. T. Sasaki and H. Takagi: Phosphorylation of a conserved Thr357 in yeast Nedd4-like ubiquitin ligase Rsp5 is involved in downregulation of the general amino acid permease Gap1. Genes Cells, 18, 459-475 (2013).
  16. T. Ogata, H. Yamagishi, K. Ukibe and H. Takagi: Construction of bottom-fermenting yeasts that overexpress the ubiquitin ligase RSP5 gene and their effects on high-gravity fermentation. J. Brew. Distill., 3, 1-5 (2012).
  17. H. Hiraishi, T. Shimada, I. Ohtsu, T. Sato and H. Takagi: The yeast ubiquitin ligase Rsp5 down-regulates the alpha subunit of nascent polypeptide-associated complex Egd2 under stress conditions. FEBS J., 276, 5287-5297 (2009).
  18. H. Hiraishi, M. Okada, I. Ohtsu and H. Takagi: Functional analysis of the yeast ubiquitin ligase Rsp5: Involvement of the ubiquitin-conjugating enzyme Ubc4 and poly-ubiquitination in ethanol-Induced down-regulation of targeted proteins. Biosci. Biotech. Biochem., 73, 2268-2273 (2009).
  19. Y. Haitani, M. Nakata, T. Sasaki, A. Uchida and H. Takagi: Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1. FEMS Yeast Res., 9, 73-86 (2009).
  20. Y. Haitani and H. Takagi: Rsp5 is required for the nuclear export of mRNA of HSF1 and MSN2/4 under stress conditions in Saccharomyces cerevisiae. Genes to Cells, 13, 105-116 (2008).
  21. M. Demae, Y. Murata, M. Hisano, Y. Haitani, J. Shima and H. Takagi: Overexpression of two transcriptional factors, Kin28 and Pog1, suppresses the stress sensitivity caused by the rsp5 mutation in Saccharomyces cerevisiae. FEMS Microbiol. Lett., 277, 70-78 (2007).
  22. H. Hiraishi, M. Mochizuki and H. Takagi: Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Biosci. Biotech. Biochem., 70, 2762-2765 (2006).
  23. Y. Haitani, H. Shimoi and H. Takagi: Rsp5 regulates expression of stress proteins via post-translational modification of Hsf1 and Msn4 in Saccharomyces cerevisiae. FEBS Lett., 580, 3433-3438 (2006).
  24. H. Takagi: Analysis and application of degradation mechanism of abnormal proteins by the yeast Rsp5 ubiquitin ligase. Report of the Noda Institute for Scientific Research, 49, 78-80 (2005).
  25. C. Hoshikawa, M. Shichiri, S. Nakamori and H. Takagi: A non-conserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins. Proc. Natl. Acad. Sci. USA, 100, 11505-11510 (2003).