78NISR RESEARCH GRANT
2004 Research Grant

Analysis and Application of Degradation Mechanism ofAbnormal Proteins by the Yeast Rsp5 Ubiquitin Ligase
Hiroshi TAKAGI
Department of Bioscience, Fukui Prefectural University
Research aims
Yeast cells are exposed to environmental stresses during
fermentations. Stress induces protein denaturation, generates abnormal proteins, and leads to growth inhibition orcell death. Therefore, stress tolerance is the key for application to industrial yeasts. We analyze the stress-tolerant
mechanism and its application in yeast based on abnormal
proteins1–9.
Rsp5 is an essential E3 ubiquitin ligase containing theHECT-domain in budding yeast Saccharomyces cerevisiae.
We previously isolated the rsp5 mutant with a single aminoacid substitution, Ala401Glu, in the third WW domain (aprotein interaction module). Interestingly, when yeast cells
were exposed to stresses such as toxic amino acid analogues, high growth temperature in a rich medium, ethanol,and oxidative treatments, the rsp5 mutant showed much
more sensitivity to these stresses than the wild-type strain.
These results suggest that Rsp5 is required for selectivedegradation of abnormal proteins generated by thesestresses, in addition to nitrogen-regulated degradation ofGap15.
In this study, focused on the degradation and repair systems of stress-induced abnormal proteins, we analyzed thefunction of Rsp5 under stress conditions.
Methods
1) Degradation system
To identify stress-induced substrates of Rsp5, we performed proteome analysis of the wild-type and the rsp5
mutant strains using comparative 2D-PAGE and mass spectrometry.
2) Repair system
To analyze the effect of Rsp5 on the expression of stressproteins, we examined the transcription of three genes
HSP42, DDR2 and HSP12, using HSE-lacZ, STRE-lacZand HSESTRE-lacZ, respectively, as reporter in the wildtype and rsp5 mutant cells under various stress conditions.

tant compared with those of the wild-type strain under highgrowth temperature in a rich medium (Fig. 1). The proteome analyses of wild-type and rsp5 mutant strains suggested a possibility that the stress-induced proteins are substrates of Rsp5. Therefore, we investigated the interactionsof Rsp5 and candidate substrates using two-hybrid assay.
The stress-induced proteins interacted with Rsp5. Our results suggest that high temperature-induced abnormal proteins interact with the third WW domain of Rsp5, and are
degraded selectively in proteasome or vacuole.
2) Repair system
The accumulation of abnormal or misfolded proteins in
the cells under stress is a serious problem. To overcome it,
two strategies can be considered: (i) to degrade the proteinsthrough ubiquitin-proteasome system or (ii) to refold theproteins by molecular chaperones including stress proteins.
In S. cerevisiae, two transcription factors, Hsf1 andMsn2/4, appear to be responsible for stress-induced geneexpression. Hsf1 binds to heat shock elements (HSEs) andMsn2/4 binds to stress response elements (STREs) found in
the promoter of many heat-inducible genes encoding stressproteins.
The b -galactosidase activities in the rsp5 mutant weresignificantly lower than that in the wild-type strain when
exposed to temperature upshift (Fig. 2), ethanol, sorbitol orheat shock treatment. Similar results were obtained from
Northern blot analysis. The rsp5 mutant fails to degrade
and to refold stress-induced abnormal proteins. Overexpression of stress proteins complemented stress hypersensi-Results1) Degradation system
The accumulation of abnormal proteins in cells is a serious problem. Rsp5 is believed to function as an ubiquitin
ligase under various stress conditions, but its substrates remain unclear. Interestingly, some proteins such as Hsp12,Hsp78, Egd2 and Sug2 were accumulated in the rsp5 mu-Fig. 1. 2D-PAGE pattern after growth at high temperature (37°C) in arich medium.

79Fig. 2.

Transcription analysis of stress proteins when exposed to temperature-upshift in the wild-type and rsp5 mutant strains.

Fig. 3.

Overexpression of HSP42, DDR2 and HSP12 in the rsp5 mutant.

tivity of the rsp5 mutant (Fig. 3). These results indicate thatRsp5 regulates the expression of stress proteins.
Conclusions
1) Degradation system
Some proteins such as Hsp12, Hsp78, Egd2 and Sug2

2) Repair system
Under stress conditions (temperature upshift, ethanol,sorbitol and heat shock), the rsp5 mutant resulted in decreased expression of stress proteins (Hsp42, Ddr2 andwere accumulated in the rsp5 mutant compared with thoseof wild-type strain under high growth temperature in a richmedium. Some proteins including Hsp12, Hsp78 and Sod1
were also accumulated under ethanol stress. Two-hybrid
assay showed the weak interaction of Rsp5 and stress-induced proteins, suggesting that these proteins are substratesof Rsp5.

Hsp12). Rsp5 may have a novel function that regulates theexpression of stress proteins through ubiquitination of two
transcription factors (Hsf1 and Msn2/4).

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References
1.

H. Takagi, K. Sakai, K. Morida and S. Nakamori: Proline accumulation by mutation or disruption of the proline oxidase gene improves
resistance to freezing and desiccation stresses in Saccharomyces
cerevisiae. FEMS Microbiol. Lett., 184, 103–108 (2000).
2. M. Shichiri, C. Hoshikawa, S. Nakamori and H. Takagi: A novel
acetyltranferase found in Saccharomyces cerevisiae S 1278b that
detoxifies a proline analogue, azetidine-2-carboxylic acid. J. Biol.
Chem., 276, 41998–42002 (2001).
3. Y. Morita, S. Nakamori and H. Takagi: Effect of proline and arginine
metabolism on freezing stress of Saccharomyces cerevisiae. J.
Biosci. Bioeng., 94, 390–394 (2002).
4. Y. Morita, S. Nakamori and H. Takagi: L-Proline accumulation andfreeze tolerance in Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding g -glutamyl kinase. Appl. Environ.
Microbiol., 69, 212–219 (2003).

5.

C. Hoshikawa, M. Shichiri, S. Nakamori and H. Takagi: A non-conserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved indegradation of Gap1 permease and stress-induced abnormal proteins.
Proc. Natl. Acad. Sci. U.S.A., 100, 11505–11510 (2003).
6. Y. Terao, S. Nakamori and H. Takagi: Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae. Appl. Environ. Microbiol., 69,6527–6532 (2003).
7. M. Nomura and H. Takagi: Role of the yeast novel acetyltransferase
Mpr1 in oxidative stress: Regulation of oxygen reactive species
caused by a toxic proline catabolism intermediate. Proc. Natl. Acad.
Sci. U.S.A., 101, 12616–12621 (2004).
8. X. Du and H. Takagi: N-Acetyltransferase Mpr1 conferred freeze tolerance in Saccharomyces cerevisiae by reducing reactive oxygenspecies. J. Biochem., in press.
9. K. Matsuura and H. Takagi: Vacuolar functions are involved instress-protective effect of intracellular proline on Saccharomyces
cerevisiae. J. Biosci. Bioeng., in press.