Stress-mediated inhibition of the classical nuclear protein import pathway and nuclear accumulation of the small GTPase Gsp1p.
Stress modifies all aspects of cellular physiology, including the targeting of macromolecules to the nucleus. To determine how distinct types of stress affect classical nuclear protein import, we followed the distribution of NLS-GFP, a reporter protein containing a classical nuclear localization sequence (NLS) fused to green fluorescent protein GFP. Nuclear accumulation of NLS-GFP requires import to be constitutively active; inhibition of import redistributes NLS-GFP throughout the nucleus and cytoplasm. In the yeast Saccharomyces cerevisiae, starvation, heat shock, ethanol and hydrogen peroxide rapidly inhibited classical nuclear import, whereas osmotic stress had no effect. To define the mechanisms underlying the inhibition of classical nuclear import, we located soluble components of the nuclear transport apparatus. Failure to accumulate NLS-GFP in the nucleus always correlated with a redistribution of the small GTPase Gsp1p. Whereas predominantly nuclear under normal conditions, Gsp1p equilibrated between nucleus and cytoplasm in cells exposed to starvation, heat, ethanol or hydrogen peroxide. Furthermore, analysis of yeast strains carrying mutations in different nuclear transport factors demonstrated a role for NTF2, PRP20 and MOG1 in establishing a Gsp1p gradient, as conditional lethal alleles of NTF2 and PRP20 or a deletion of MOG1 prevented Gsp1p nuclear accumulation. On the basis of these results, we now propose that certain types of stress release Gsp1p from its nuclear anchors, thereby promoting a collapse of the nucleocytoplasmic Gsp1p gradient and inhibiting classical nuclear protein import.
Stochaj
U
U
Department of Physiology, McGill University, Montreal, PQ, Canada, H3G 1Y6. stochaj@med.mcgill.ca
Rassadi
R
R
Chiu
J
J
D001692:Biological Transport / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D008298:Maleates / Q000494:pharmacology; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology; D020931:ran GTP-Binding Protein / Q000378:metabolism
D005656:Fungal Proteins; C080324:GSP1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D008298:Maleates; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000431:Ethanol; D012965:Sodium Chloride; D013012:Sorbitol; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D020559:Monomeric GTP-Binding Proteins; D020931:ran GTP-Binding Protein; C014476:diethyl maleate