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.

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or ␤-galactosidase to nuclei. To determine whether nuclear accumulation of Star-␤-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single ␤-importin gene. With this assay we have identified Nmd5p as a ␤-importin required to concentrate Star-␤-galactosidase in nuclei when cells enter stationary phase.In eukaryotic cells, DNA replication and RNA synthesis take place in the nucleus, whereas protein synthesis occurs in the cytoplasm. Proper communication between these processes depends on the transport of soluble factors between both compartments. Nucleocytoplasmic trafficking requires that proteins cross the nuclear envelope. To do so, proteins travel through nuclear pore complexes, large specialized structures that span both the inner and outer nuclear membrane. Proteins that are smaller than 40 -60 kDa can diffuse through nuclear pore complexes without the requirement of energy. In contrast, macromolecules with a molecular mass larger than 40 -60 kDa enter the nucleus via active transport (reviewed in Refs. 1 and 2).

Cytoplasmic proteins of the hsp70/hsc70 family redistribute in cells that have been exposed to stress. As such, the hsp70 Ssa4p of the budding yeast S. cerevisiae accumulates in nuclei when cells are treated with ethanol, whereas classical nuclear import is inhibited under these conditions. The N-terminal domain of Ssa4p, which is lacking a classical NLS, mediates nuclear accumulation upon ethanol exposure. Concentration of the Ssa4p N-terminal segment in nuclei is reversible, as the protein relocates to the cytoplasm when cells recover. Mutant analysis demonstrates that the small GTPase Gsp1p and GTPase-modulating factors are required to accumulate Ssa4p in nuclei upon ethanol stress. Moreover, we have identified the importin-beta family member Nmd5p as the nuclear carrier for Ssa4p. Ethanol treatment significantly increases the formation of import complexes containing Nmd5p and the N-terminal Ssa4p domain. Likewise, docking of the carrier Nmd5p at the nuclear pore is enhanced by ethanol. Furthermore, we show that the stressed-induced nuclear accumulation of Ssa4p depends on signaling through protein kinase C and requires sensors of the cell integrity pathway.

The nuclear envelope is crucial for the functional organization of the nucleus. Lamin B receptor (LBR) and several lamina-associated proteins (LAPs), residing in the inner membrane, provide attachment sites for chromatin and the nuclear lamina. LAPs and LAP-related proteins are members of a growing family of proteins, whose genes are expressed in a tissue and development specific manner, opening the opportunity for a complex regulation of membrane-chromatin and membrane-lamina interactions. Post-translational modifications of LBR and LAPs are likely to modulate their binding to lamins and chromatin, interactions that need to be dynamic to accommodate nuclear growth in interphase and nuclear envelope disassembly in mitosis. Accumulation of proteins in the inner nuclear membrane is believed to depend on their retention mediated by the interaction with nuclear components such as chromatin and lamins.z 1998 Federation of European Biochemical Societies.