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First published online 27 July 2004
doi: 10.1242/jcs.01296

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Stress response in yeast mRNA export factor: reversible changes in Rat8p localization are caused by ethanol stress but not heat shock

Laboratory of Molecular Microbiology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

View larger version (52K): [in a new window]   Fig. 1. Effects of ethanol stress on the localization of nucleoporins and export of mRNA in yeast (Saccharomyces cerevisiae). (A) The localization of nucleoporins was observed by immunofluorescence using mAb414. Cells in exponential phase were treated with ethanol (10% v/v) for 15 minutes. (B) Intracellular localization of bulk poly(A)+ mRNA was analyzed by in situ hybridization using a DIG-labeled oligo(dT)50 probe. Cells were treated with ethanol (10% v/v) for 15 minutes or heat shock (42°C) for 60 minutes. Nuclear DNA was stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (lower panels). (C) Intracellular localization of SSA4 mRNA was analyzed by in situ hybridization using Cy3-labeled SSA4 probes. Cells were treated with ethanol (10% v/v) for 15 minutes or heat shock (42°C) for 60 minutes. Nuclear DNA was stained with DAPI (lower panels).

View larger version (80K): [in a new window]   Fig. 2. Change in the localization of mRNA export factors. The localization of mRNA export factors was monitored in living yeast cells expressing GFP fusion proteins. Cells in exponential phase were treated with ethanol (10% v/v) for 5 minutes or with heat shock stress (42°C) for 60 minutes. Rat7p-GFP and GFP-Rat8p fluorescence were visualized before ethanol treatment (w/o stress) after ethanol treatment for 30 minutes (10% EtOH) and after heat shock (42°C). Lower panels are phase-contrast images of the same cells pictured above.

View larger version (92K): [in a new window]   Fig. 3. Reversible change in the localization of GFP-Rat8p. Yeast cells in exponential phase were treated with 10% ethanol for 30 minutes, collected, and transferred to fresh SD medium without ethanol. GFP-Rat8p fluorescence was visualized before ethanol treatment (w/o stress) after ethanol treatment for 30 minutes (10% EtOH) and 5 minutes after the shift to fresh SD medium. Cells at each stage were also fixed for in situ hybridization using Cy3-labeled SSA4 probes to detect poly(A)+ RNA and DAPI staining.

View larger version (89K): [in a new window]   Fig. 4. Concentration-dependent effects of ethanol on the localization of Rat8p and export of bulk poly(A)+ mRNA. Yeast cells in exponential phase were treated with ethanol (6-9% v/v) for 15 minutes and the localization of GFP-Rat8p was monitored. Cells at each ethanol concentration were also fixed for in situ hybridization using Cy3-labeled SSA4 probes to detect poly(A)+ RNA and DAPI staining.

View larger version (59K): [in a new window]   Fig. 5. Function of Xpo1p/Crm1p under ethanol stressed conditions. (A) GFP-Rat8p accumulates in the yeast cell nucleus at the non-permissive temperature of 37°C in xpo1-1 cells. (B) Yeast cells expressing NLS-NES-GFP in exponential phase at 28°C were treated with ethanol (6 or 10% v/v) or heat shock (42°C) for 15 minutes, and then the cellular localization of NLS-NES-GFP-fusion protein was monitored and compared to non-stressed cells (w/o stress). (C,D) Cells expressing GFP-Yap1p (C) or GFP-Yap1p-cm46A5 (D) in exponential phase were treated with stress (10% ethanol, heat shock at 42°C or 0.4 mM H2O2) for 15 minutes and the cellular localization of GFP-fusion proteins was monitored.

View larger version (78K): [in a new window]   Fig. 6. Overexpression of Rat8p attenuated the blocking of export of bulk poly(A)+ mRNA in ethanol stressed cells. (A) Yeast cells overexpressing GFP-Rat8p (W303-1A with pRS426-GFP-RAT8) were treated with 10% ethanol for 15 minutes. GFP-Rat8p fluorescence was detected before (w/o stress) and after (10% EtOH) treatment. (B) Cells overexpressing Rat8p (W303-1A with pRS426-RAT8) were treated with 10% ethanol for 15 minutes and localization of bulk poly(A)+ mRNA was monitored before and after treatment. Cells carrying the empty vector (pRS426) were used as a control. Cell nuclei were stained with DAPI.