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Stress-mediated inhibition of the classical nuclear protein import pathway and nuclear accumulation of the small GTPase Gsp1p¹

Department of Physiology, McGill University, Montreal, PQ, Canada, H3G 1Y6

²Correspondence: Department of Physiology, McGill University, Montreal, PQ, Canada, H3G 1Y6. E-mail: stochaj{at}med.mcgill.ca

SPECIFIC AIMS

In the yeast Saccharomyces cerevisiae, we have analyzed the effect of different types of stress on classical nuclear protein import and nuclear accumulation of the RNA binding protein Npl3p. Furthermore, it was determined whether these stresses alter the cellular distribution of soluble nuclear transport factors, such as the small GTPase Gsp1p. As Gsp1p plays a key role in nuclear trafficking, we have studied a variety of yeast mutants to identify factors involved in Gsp1p nuclear accumulation.

PRINCIPAL FINDINGS

1. Classical nuclear protein import is inhibited upon starvation, exposure to ethanol, heat shock, and oxidative stress
The 42 kDa fluorescent reporter protein NLS-GFP, which contains a classical nuclear localization sequence (NLS) but lacks nuclear retention or export signals, was used to determine the effects of stress on nuclear protein import. Due to its small size, NLS-GFP diffuses across the nuclear envelope; its accumulation in the nucleus requires protein import to be constitutively active. Exponentially growing yeast cells efficiently imported NLS-GFP into nuclei (Fig. 1A, b ). In stationary phase cells, which are depleted for nutrients, the reporter protein distributed throughout nucleus and cytoplasm (Fig. 1A, d ). Ethanol or mild heat shock (37°C) equilibrated NLS-GFP between nucleus and cytoplasm. After 10 min treatment with ethanol, NLS-GFP distributed uniformly throughout both compartments (Fig. 1A, f ), whereas a 30 min heat shock at 37°C was required for nucleocytoplasmic equilibration of the reporter protein (Fig. 1A, h, j ). After prolonged incubation at 37°C, cells adapted to heat stress and NLS-GFP reaccumulated in nuclei (Fig. 1A, l ). Nuclear accumulation of NLS-GFP was also sensitive to hydrogen peroxide and diethyl maleate, with hydrogen peroxide being particularly effective (Fig. 1B, b, d ). By contrast, short-term exposure to osmotic stress or incubation with cycloheximide did not prevent NLS-GFP nuclear accumulation (Fig. 1B, f, h, j ).

View larger version (33K): [in this window] [in a new window]   Figure 1. A) Effect of starvation, ethanol, and heat stress on the nuclear accumulation of NLS-GFP. The reporter protein NLS-GFP was located in yeast strain RS453. Cells growing exponentially (a, b, e–l), in stationary phase (c, d), and exposed to ethanol (10%, 10 min; e, f) or heat (g–l) were analyzed. Nuclei were detected with 4',6-diamidino-2-phenylindole (DAPI, a, c, e, g, i, k); NLS-GFP was located by its green fluorescence (b, d, f, h, j, l). B) Effect of oxidative stress, osmotic stress, and cycloheximide on nuclear import of NLS-GFP. Exponentially growing yeast cells expressing the NLS-GFP gene were incubated with hydrogen peroxide (2 mM, 10 min; a, b), diethyl maleate (2 mM, 2 h; c, d), NaCl (0.4 M, 10 min; e, f), sorbitol (1 M, 10 min; g, h), or cycloheximide (100 µg/ml, 4 h; i, j). Staining of the DNA with DAPI (a, c, e, g, i) and green fluorescence (b, d, f, h, j) is shown.

2. Starvation, heat stress, and sorbitol relocate Npl3p, an mRNA binding protein with a nonclassical NLS, to the cytoplasm
Npl3p, a shuttling RNA binding protein with a nonclassical NLS, is concentrated in nuclei, where it associates with mRNAs to be exported to the cytoplasm. Localization of Npl3p is regulated by nuclear import, nuclear export, and nuclear retention, as Npl3p interacts with mRNA and possibly other components of the RNP–export complex. Severe heat shock, starvation, or incubation with sorbitol were the only treatments that significantly increased Npl3p concentrations in the cytoplasm.

3. Stress alters the cellular localization of the small GTPase Gsp1p
Classical nuclear protein import requires nucleocytoplasmic gradients of the small GTPase Gsp1p and its modulating factor Prp20p. When analyzed in unstressed cells, Gsp1p and Prp20p were mostly associated with nuclei, and a portion of Gsp1p and Prp20p was also detected in the cytoplasm. Srp1p, the subunit of the classical NLS-receptor, is nuclear as well as cytoplasmic under normal conditions, being somewhat concentrated at nuclei.

Starvation, ethanol, heat, and hydrogen peroxide altered the cellular distribution of Gsp1p. A 10 min heat shock at 37°C equilibrated Gsp1p between nucleus and cytoplasm in approximately half of the cells, similar to the relocation of NLS-GFP (Fig. 1A, h ). Starvation or incubation with ethanol and hydrogen peroxide affected Gsp1p more prominently; the GTPase distributed uniformly throughout nucleus and cytoplasm. Osmotic stress was not effective to relocate Gsp1p. Thus, starvation, ethanol, heat shock, and hydrogen peroxide, which efficiently inhibited classical nuclear protein import, also altered the nucleocytoplasmic gradient of Gsp1p. By contrast, changes in the localization of Srp1p or Prp20p did not correlate with the inhibition of classical nuclear transport.

4. Ntf2p, Prp20p, and Mog1p regulate nuclear accumulation of the small GTPase Gsp1p
Various components interact with Gsp1p, including Ntf2p, a Gsp1p-GDP binding protein, the nucleotide exchange factor Prp20p, the GTPase-activating protein Rna1p, members of the importin-ß family, and the nuclear protein Mog1p, which specifically binds Gsp1p-GTP. Several conditional lethal alleles of NTF2, PRP20, RNA1, GTR1 (encoding a GTPase), or members of the importin-ß family and a deletion of MOG1 are temperature sensitive for classical and nonclassical nuclear transport. Cytoplasmic hsp70s and several nucleoporins also play a role in nuclear import. Although heat shock destroys the Gsp1p gradient, wild-type cells adapt, and Gsp1p concentrated again in nuclei 1 h after transfer to 37°C. Incubation at 37°C for up to 3 h did not affect Gsp1p distribution in wild-type cells. Similarly, NLS-GFP reaccumulated in wild-type cells that were kept at 37°C for 1 h (Fig. 1A, l ). We have used this assay to test temperature-sensitive yeast strains for their capacity to rebuild the nucleocytoplasmic Gsp1p gradient when incubated at 37°C (summarized in Table 1 ). After 3 h at the nonpermissive temperature, the Gsp1p gradient remained collapsed in about 70% of the yeast cells carrying the ntf2–1 or ntf2–2 allele, but not in wild-type cells.

The GTP/GDP exchange factor Prp20p resides in the nucleus where it generates Gsp1p-GTP. However, the mutant protein prp20–1p mislocalizes under nonpermissive conditions, and mistargeting of prp20–1p to the cytoplasm correlated with the nucleocytoplasmic equilibration of Gsp1p. In addition, mog1 cells showed increasing concentrations of Gsp1p in the cytoplasm when kept at 37°C.

5. Mutations in several nuclear transport factors do not prevent the formation of a Gsp1p gradient
We have tested additional strains with conditional lethal alleles of genes involved in nuclear transport or organization. However, none of the mutations abolished nuclear accumulation of Gsp1p (Table 1) . Thus, importin-ß, various members of the importin-ß family, several nucleoporins, cytoplasmic hsp70s, the GTPase Gtr1p, the nucleolar protein Nop1p, and Rip1p were not essential to generate a nucleocytoplasmic gradient of Gsp1p.

CONCLUSIONS

We have demonstrated that starvation, ethanol, heat, or oxidants equilibrate the classical transport substrate NLS-GFP between nucleus and cytoplasm. This can be attributed specifically to an inhibition of nuclear import, as localization of the fluorescent GFP-tag was not affected under these conditions. Moreover, nuclear import was not prevented by an unspecific obstruction of nuclear pore complexes, since NLS-GFP could still exit the nucleus by diffusion.

The effect of stress on the RNA binding protein Npl3p is more complex, as its distribution depends on nuclear import, nuclear export, and nuclear retention. Only severe heat shock, starvation, and some forms of osmotic stress were found to prevent nuclear retention as well as nuclear import of Npl3p.

Several soluble components and nucleoporins are essential for classical nuclear protein transport. In particular, a nucleocytoplasmic gradient of Gsp1p was proposed to be required, with high concentrations of Gsp1p in nuclei. Nuclear Gsp1p is believed to be associated with GTP, whereas Gsp1p-GDP is predominantly cytoplasmic. As demonstrated by us, inhibition of classical nuclear protein import corresponds to the collapse of the nucleocytoplasmic gradient of Gsp1p. We therefore propose a redistribution of Gsp1p as the mechanism that underlies stress-dependent inactivation of classical nuclear protein import (Fig. 2 ).

View larger version (13K): [in this window] [in a new window]   Figure 2. Schematic diagram describing the inhibition of classical nuclear protein import in yeast. Certain forms of stress change the nucleocytoplasmic gradient of Gsplp. The same is observed if cells carry one of the conditional lethal alleles indicated and are grown under nonpermissive conditions. As a consequence of stress or the mutation, the Gsplp gradient collapses and classical nuclear protein import is inhibited.

In addition, we have identified several factors that are involved in the formation of a nucleocytoplasmic Gsp1p gradient. Thus, temperature-sensitive alleles of NTF2, PRP20, or a deletion of MOG1 affected Gsp1p accumulation in nuclei. Deletions or mutations in single members of the importin-ß family did not abolish the formation of a Gsp1p gradient, consistent with the idea that several importin-ß proteins can anchor Gsp1p in the nucleus. Elimination of one of the anchors does not suffice to collapse the Gsp1p gradient, as other importin-ß proteins will provide nuclear binding sites for Gsp1p. Furthermore, many of the mutations that affect classical or nonclassical nuclear protein transport did not abolish the formation of a nucleocytoplasmic Gsp1p gradient, demonstrating that they inhibit nuclear trafficking via different mechanisms.

In summary, our studies have defined components in S. cerevisiae that regulate the generation and/or maintenance of the Gsp1p gradient and thereby modulate nuclear protein transport. Our data contribute to the understanding of stress-induced inhibition of classical nuclear import. Gsp1p redistributes in response to certain types of stress that are also known to deplete intracellular ATP pools. As a model, we propose that ATP depletion reduces the GTP/GDP ratio, which will favor the formation of Gsp1p-GDP. This will liberate Gsp1p from nuclear anchors that specifically bind Gsp1p-GTP. After release, Gsp1p will diffuse across the nuclear envelope, finally equilibrating between nucleus and cytoplasm. Our model implies that stress-induced energy depletion triggers a collapse of the Gsp1p gradient, thereby inhibiting classical nuclear protein import.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-0751fje To cite this article, use (September 8, 2000) FASEB J. 10.1096/fj.99-99-0751fje

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