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Regulated nuclear accumulation of the yeast hsp70 Ssa4p in ethanol-stressed cells is mediated by the N-terminal domain, requires the nuclear carrier Nmd5p and protein kinase C¹

Physiology Department, McGill University, Montreal, PQ, Canada

²Correspondence: Physiology Department, McGill University, 3655 Promenade Sir William Osler, Montreal, PQ, Canada H3G 1Y6. E-mail: ursula.stochaj{at}mcgill.ca

SPECIFIC AIMS

Cytoplasmic proteins of the heat shock protein (hsp)70 family, such as Ssa4p in Saccharomyces cerevisiae, accumulate in the nuclei in response to certain forms of stress. With the experiments described here, we have defined the signal and the molecular mechanisms that mediate the nuclear concentration of Ssa4p in response to ethanol exposure.

PRINCIPAL FINDINGS

1. The cytoplasmic hsp70 Ssa4p specifically accumulates in the nuclei upon incubation with ethanol
Various types of stress may alter the nucleocytoplasmic distribution of proteins. Ssa4p, cytoplasmic and nuclear under normal physiological conditions, accumulates in the nuclei when cells have been treated with ethanol. By contrast, the classical nuclear transport pathway is inhibited by ethanol (Fig. 1 A, C). Unlike ethanol treatment, osmotic or oxidative stress fails to concentrate Ssa4p in the nuclei.

View larger version (58K): [in this window] [in a new window]   Figure 1. Stress-induced relocation of the cytoplasmic hsp70 Ssa4p and NLS–green fluorescent protein (GFP). Wild-type yeast cells synthesizing Ssa4p fusion proteins or NLS–GFP were grown overnight in drop-out medium containing 2% galactose. In fixed cells, DNA was visualized with 4',6-diamidino-2-phenylindole (DAPI), and fusion proteins were localized by fluorescence microscopy. The experiment was performed with (A) GFP–Ssa4p(16–642), (B) Ssa4p(1–236)–GFP, and (C) NLS–GFP. GFP-containing reporter proteins were located in control cells (b) or after 10 min exposure to 10% ethanol (EtOH; d), 0.4 M sodium chloride (NaCl; f), or 0.3 mM hydrogen peroxide (h).

2. The N-terminal domain of Ssa4p is sufficient to concentrate Ssa4p in the nuclei
Ssa4p contains a segment with clusters of basic amino acid residues that is similar to a classical nuclear localization sequence (cNLS). However, this potential NLS is not required for ethanol-induced nuclear localization. An N-terminal segment of 236 amino acid residues of Ssa4p fused to GFP, termed Ssa4p(1–236)–GFP, is lacking the potential cNLS but mediates the nuclear accumulation of the non-nuclear reporter protein GFP (Fig. 1B ). These results are in line with the hypothesis that a nonclassical signal and transport route is used for Ssa4p nuclear import.

3. Different targeting signals are used to accumulate Ssa4p in ethanol stressed and starving cells
Our previous studies have shown that a short, hydrophobic sequence in the N-terminal domain of Ssa4p, termed Star (for starvation), promotes targeting to the nucleus of starving cells when fused to ß-galactosidase. By contrast, Star–ß-galactosidase does not concentrate in the nuclei in response to ethanol treatment. These results suggest that Ssa4p contains more than one signal for nuclear import and that distinct signals are used when cells have been exposed to different forms of stress.

4. Nuclear accumulation of Ssa4p–GFP is reversible
The concentration of Ssa4p in the nuclei of stressed cells is only transient. Upon removal of the stressor ethanol, Ssa4p(1–236)–GFP relocates to the cytoplasm, suggesting that this protein shuttles between both compartments.

5. Nuclear accumulation of Ssa4p in ethanol-treated cells requires the Ran/Gsp1p GTPase cycle
Different conditional lethal mutants deficient in distinct nuclear transport factors were analyzed for their capacity to import the Ssa4p N-terminal domain into nuclei upon exposure to ethanol. These studies revealed that Ran/Gsp1p and the GTPase-interacting factors Rna1p (GTPase-activating protein) and Prp20p (guanine nucleotide exchange factor) are necessary for Ssa4p nuclear import in stressed cells.

6. Classical nuclear import is not required to accumulate Ssa4p in the nuclei of stressed cells
Classical nuclear import relies on the Ran GTPase system and the dimeric import receptor importin-/importin-ß. This import route is involved in nucleartrafficking of a wide variety of substrates that contain a cNLS. The classical nuclear import pathway is inhibited when cells have been treated with ethanol, making transport of Ssa4p(1–236)–GFP via importin-/importin-ß unlikely. In line with this hypothesis, we demonstrate that Srp1p, yeast importin-, is not essential to concentrate the reporter protein in the nuclei of ethanol-stressed cells.

7. The nuclear carrier Nmd5p concentrates Ssa4p in the nuclei after incubation with ethanol
The requirement of the Ran/Gsp1p GTPase cycle for Ssa4p nuclear accumulation suggested that a member of the importin-ß family of carriers plays a role in Ssa4p nuclear import. Indeed, the analysis of mutants inactivated for different importin-ß genes shows that the carrier Nmd5p promotes the nonclassical nuclear accumulation of Ssa4p and its N-terminal domain in ethanol-stressed cells (Fig. 2 A–C). As such, various mutant strains that contain the wild-type NMD5 gene rapidly concentrate Ssa4p(1–236)–GFP in the nuclei. By contrast, mutant cells carrying a knockout of NMD5 fail to accumulate Ssa4p in the nuclei (Fig. 2A-C ).

View larger version (60K): [in this window] [in a new window]   Figure 2. Ethanol-induced nuclear accumulation of Ssa4p(1–236) requires the carrier Nmd5p. (A)Yeast strains carrying a mutation in one of the importin-ß genes were analyzed for the nuclear accumulation of Ssa4p(1–236) in response to ethanol stress. The distribution of Ssa4p(1–236)–GFP is shown for strains nmd5::TRP1, sxm1::TRP1, and lph2::TRP1. Control and ethanol-treated cells were analyzed in parallel. (B) GFP–Ssa4p(16–642) was located in nmd5::TRP1 control and ethanol-stressed cells as indicated. (C) Ethanol-induced nuclear accumulation of Ssa4p(1–236)–GFP in mutant yeast strains deleted for one of the importin-ß genes. Mutant cells synthesizing Ssa4p(1–236)–GFP were stressed with 10% ethanol for the time indicated. Cells were fixed, and nuclear accumulation (N>C) of the fusion protein was monitored by fluorescence microscopy. Results were observed at least three times. Nuclear accumulation of Ssa4p(1–236)–GFP was determined at the times indicated, 100 cells were scored in each independent experiment for every time point, and means are depicted in the figure.

8. The formation of Ssa4p(1–236)–GFP/Nmd5p import complexes is increased by ethanol
The first step of nuclear import is the generation of the cargo/carrier complex. Ssa4p(1–236)–GFP associates with Nmd5p, and this interaction can be dissociated by Gsp1p–GTP, indicating that Ssa4p(1–236)–GFP and Nmd5p are together part of a bona fide import complex. Although Ssa4p(1–236)–GFP can bind to Nmd5p in unstressed cells, the formation of Ssa4p(1–236)–GFP/Nmd5p import complexes is significantly increased by exposure to ethanol.

9. Binding of the carrier Nmd5p to nuclear pore complexes (NPC) is stimulated by ethanol
Following the formation of import complexes, nuclear carriers will dock at the nuclear pore to promote the translocation of its cargo. Nmd5p binding to nuclear pore complexes is regulated by ethanol, and it is significantly enhanced in cells that have been exposed to this alcohol.

10. Ethanol-induced concentration of Ssa4p in the nuclei requires protein kinase C (Pkc1p) and sensors of the cell integrity signaling pathway
Nuclear accumulation of Ssa4p(1–236)–GFP was tested in strains deleted for the single gene, which encodes Pkc1p in budding yeast. This mutant is unable to concentrate the reporter protein in the nuclei when cells have been exposed to ethanol. MID2 and WSC1 code for proteins located in the plasma membrane; they act as sensors of cell integrity and can induce the activation of Pkc1p. The deletion of MID2 or WSC1 significantly reduces the nuclear accumulation of Ssa4p(1–236)–GFP in stressed cells, suggesting a link between Pkc1p activation and nuclear import of Ssa4p.

CONCLUSIONS AND SIGNIFICANCE

The appropriate response to stress is crucial to cell survival and recovery from various insults. As such, the nuclear accumulation of proteins of the hsp70/hsc70 family plays a critical role in the repair of stress-induced injuries. Nuclear proteins may be particularly vulnerable to damage by ethanol, therefore requiring the nuclear accumulation of heat shock protein to restore cellular functions. Different mechanisms may induce the concentration of a protein in the nucleus. These could include an increase in nuclear import, inhibition of nuclear export, as well as changes in nuclear or cytoplasmic retention. Our studies suggest that the up-regulation of Ssa4p nuclear import contributes to its concentration in the nuclei. We show that a nonclassical transport route which requires Nmd5p, a member of the importin-ß family of nuclear carriers, mediates Ssa4p nuclear import in ethanol-treated cells. For this transport pathway, we have identified two distinct steps that are modulated by stress. First, the formation of import complexes containing Ssa4p(1–236)–GFP and Nmd5p is increased in ethanol-treated cells. Second, docking of the carrier Nmd5p at the nuclear pore complex is enhanced when cells have been incubated with ethanol. Both changes in Ssa4p nuclear trafficking can be expected to promote the concentration of the heat shock protein in the nuclei (summarized in Fig. 3 ). The regulation of hsp70 nuclear transport on different levels of the import reaction may help cells to fine-tune the efficiency of the nuclear accumulation according to the severity of stress and the physiological requirements of the cell.

View larger version (20K): [in this window] [in a new window]   Figure 3. Concentration of Ssa4p in the nuclei upon exposure to ethanol stress. Simplified model for the nuclear accumulation of the hsp70 Ssa4p in cells that have been treated with ethanol. See text for details.

In addition to identifying the nuclear transport pathway for Ssa4p, our results show that Pkc1p plays an essential role in nuclear trafficking of the heat shock protein. Moreover, Mid2p and Wsc1p, sensors of the cell-integrity pathway, which are known to be upstream activators of Pkc1p, are necessary to concentrate Ssa4p(1–236)–GFP in the nuclei of ethanol-treated cells. We propose that Mid2p and Wsc1p sense changes in the membrane fluidity induced by ethanol, leading to the activation of Pkc1p and ultimately to the nuclear accumulation of Ssa4p. Future experiments will have to define the molecular reactions that link Pkc1p activation to the concentration of Ssa4p in the nuclei.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0947fje

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