Starvation Promotes Nuclear Accumulation of the hsp70 Ssa4p in Yeast Cells

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 β-galactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay we have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei when cells enter stationary phase. 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 β-galactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay we have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei when cells enter stationary phase. nuclear localization sequence E. coliβ-galactosidase 4′,6-diamidino-2-phenylindole A. victoria green fluorescent protein protein sequence inducing nuclear targeting upon starvation 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. 1Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar and 2Stochaj U. Rother K.L. BioEssays. 1999; 21: 579-589Crossref Scopus (26) Google Scholar). A variety of pathways mediate the nuclear accumulation of proteins too large to diffuse into the nucleus (reviewed in Refs. 1Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar and 2Stochaj U. Rother K.L. BioEssays. 1999; 21: 579-589Crossref Scopus (26) Google Scholar). In most cases, nuclear trafficking depends on specific carrier molecules termed importins. In the yeast Saccharomyces cerevisiae 14 members of the β-importin family have been identified (reviewed in Ref. 3Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), three of which are essential for cell viability. These carriers are involved in nuclear export or import of proteins and RNA. At present, classical nuclear protein transport is the pathway understood best. This transport route requires an adaptor protein, α-importin (Srp1p in yeast) that links the nuclear cargo to β-importin (Rsl1p in yeast). Like other forms of nuclear transport, classical nuclear protein import requires energy, soluble transport factors, and nuclear localization sequences (NLSs),1 specialized signals that target proteins into the nucleus. In classical nuclear transport, these signals can be of two types: monopartite or bipartite. Monopartite signals are simple stretches of basic amino acids, whereas bipartite NLSs contain two clusters of basic amino acids separated by a spacer region (reviewed in Ref. 4Dingwall C. Laskey R.A. Trends Biol. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1705) Google Scholar). In addition to the well characterized classical nuclear import route, a variety of nonclassical pathways have been described that depend on other members of the β-importin family of carriers (3Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Frequently, nonclassical transport pathways are mediated by NLSs distinct from the simple or bipartite type, and such nonclassical NLSs do not share a consensus sequence. Examples of proteins containing a nonclassical NLS include the mammalian protein hnRNPA1 and the yeast protein Npl3p (1Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar,2Stochaj U. Rother K.L. BioEssays. 1999; 21: 579-589Crossref Scopus (26) Google Scholar). In general, proteins bearing a nonclassical NLS associate directly with a specific β-importin for targeting to the nucleus, without the requirement of an adaptor. Thus, β-importins have been shown to play a role in nuclear import of ribosomal proteins, transcription factors, or RNA-binding proteins (3Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Of particular interest are β-importins involved in trafficking of proteins that regulate the response to stress or changes in nutrient availability. For instance, Nmd5p mediates nuclear import of the mitogen-activated protein kinase Hog1p upon exposure to osmotic stress, and nuclear import is triggered by Hog1p phosphorylation (5Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5506-5614Crossref Scopus (345) Google Scholar). Another example of regulated nucleocytoplasmic trafficking is Mig1p, a transcription factor promoting glucose-mediated repression of several genes (6DeVit M.J. Johnston M. Curr. Biol. 1999; 9: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Removal of glucose from the growth medium induces Mig1p phosphorylation via Snf1p. Phosphorylated Mig1p is then recognized by the β-importin Msn5p and subsequently exported to the cytoplasm (6DeVit M.J. Johnston M. Curr. Biol. 1999; 9: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). During logarithmic growth, cells deplete their medium for nutrients and enter stationary phase. Under these conditions, a variety of genes modify their expression levels. Nutrient depletion represents a specific form of stress that inhibits classical nuclear transport and can also affect nonclassical transport pathways (7Stochaj, U., Rassadi, R., and Chiu, J. (September 8, 2000) FASEB J. 10.1096/fj.99-99-0751fje.Google Scholar). Moreover, various proteins regulating the response to changing nutrient concentrations, such as Mig1p, may alter their distribution between nucleus and cytoplasm (reviewed in Ref. 8Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar). Different forms of stress alter the cellular physiology and may damage cells. To survive and recover from stress-induced injury, cells need to activate specialized survival and repair mechanisms. In particular, heat shock proteins of the hsp70/hsc70 family play an essential role in these processes. Hsp70/hsc70s shuttle between nucleus and cytoplasm (9Mandell R.B. Feldherr C.M. J. Cell Biol. 1990; 111: 1775-1783Crossref PubMed Scopus (99) Google Scholar) and accumulate in nuclei upon exposure to heat stress. Unlike heat shock, however, the effect of starvation on hsp70 localization has not been studied in detail. The yeast S. cerevisiae contains six members of the cytosolic hsp70 family. Cytosolic hsp70s are divided into two subfamilies: four SSA-encoded gene products (Ssa1p to Ssa4p) and two SSB gene products (Ssb1p and Ssb2p) (reviewed in Ref. 10Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 31-52Google Scholar). The SSA4 gene is particularly interesting as its expression is highly induced upon stress (10Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 31-52Google Scholar) and up-regulated during diauxic shift. Proteins of the SSA family are located in the cytoplasm and the nucleus, because they shuttle between both compartments under normal conditions. Unlike gene products of theSSA group, Ssb1p and Ssb2p are located predominantly in the cytoplasm. Ssb1p is prevented from nuclear accumulation due to the presence of a nuclear export signal in its N-terminal domain (11Shulga N. James P. Craig E.A. Goldfarb D.S. J. Biol. Chem. 1999; 274: 16501-16507Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It has not been analyzed previously whether nutrient depletion affects the localization of hsp70s. We have addressed this problem by generating fusions containing Ssa4p and GFP (Aequorea victoria green fluorescent protein). We now show that upon starvation GFP fusion proteins carrying Ssa4p or its N-terminal domain accumulate in nuclei. We have identified a short hydrophobic segment in this N-terminal portion, referred to as the Star sequence, which is sufficient to direct non-nuclear reporter proteins to nuclei when cells are entering stationary phase. Moreover, we demonstrate that the β-importin Nmd5p is required to import a fusion between the Star sequence and β-galactosidase into nuclei of early stationary phase cells. Yeast strain RS453 (ade2 ADE3 leu2 ura3 trp1 his3), provided by V. Doye (Paris), was used as wild type strain. Mutant yeast strains were provided by G. Schlenstedt (Hamburg, Germany). Upon transformation, cells were grown in synthetic complete medium lacking uracil or leucine (dropout medium), depending on the selectable marker introduced by the plasmid. Cells were kept at room temperature unless indicated otherwise. Expression of genes controlled by the GAL1 orGALS promoter was induced with 2% galactose as a carbon source. For data shown in Fig. 2, cells were transferred for 6 h into fresh glucose-containing medium. The following inhibitors were added as indicated in the Fig. 2: 2 mm hydrogen peroxide (American Chemicals, Montreal), 2 mm diethyl maleate (Sigma), or 100 μg/ml cycloheximide (Sigma). All plasmid constructions were carried out in Escherichia coli strain XL1-Blue. The correctness of constructs was verified by sequencing with the Sequenase 2.0 kit (U. S. Biochemical Corp.). To generate fusions between GFP and SSA4, the GFPmut1 allele was used. GFPmut1 encodes a protein with ∼35 times the fluorescence intensity of the wild type (12Cormack B.P. Valdivia R.H. Falkow S. Gene. 1996; 173: 33-38Crossref PubMed Scopus (2474) Google Scholar). Plasmid pGAD-GFP encodes NLS-GFP, a fusion protein that carries the classical SV40-NLS (13Shulga N. Roberts P. Gu Z. Spitz L. Tabb M.M. Nomura M. Goldfarb D.S. J. Cell Biol. 1996; 135: 329-339Crossref PubMed Scopus (186) Google Scholar). pGAD-GFP was kindly provided by Dr. D. Goldfarb (Rochester, NY). For GFP-Ssa4p-(16–642), aNotI-linker (12-mer, New England Biolabs, Mississauga, ON) was inserted into the unique NlaIII site of SSA4, thereby generating plasmid p930. Plasmid p930 was cut withNotI, and the GFP-coding sequence was fused in-frame to codon 16 of SSA4; the fusion gene encodes GFP-Ssa4p-(16–642). For expression in yeast, the centromeric plasmid, which carries the URA3 selectable marker, was used as a vector. Expression of the gene fusions inserted into this plasmid is regulated by the GAL1 promoter. For of GFP containing a at the S. U. Cell Biol. 1999; PubMed Google Scholar) was fused to amino to of by an To generate fusions between the Star sequence and by the Star sequence were fused to the of the β-galactosidase gene. To green fluorescent protein was fused in-frame to the of For expression in yeast, the Star-β-galactosidase gene was into centromeric with or URA3 as a selectable Yeast cells were as described previously S. U. Cell Biol. 1999; PubMed Google Scholar) and on containing 2% reporter proteins were located by fluorescence as previously described S. U. Cell Biol. 1999; PubMed Google Scholar). proteins carrying β-galactosidase were located with as in Ref. U. Cell Biol. 1996; PubMed Scopus Google To and Star-β-galactosidase cells were grown in synthetic complete medium lacking both uracil and leucine and containing 2% Cells were and with β-galactosidase and GFP were with and to DNA was with 4′,6-diamidino-2-phenylindole and were in of GFP and members of the SSA family of proteins are to their targeting the cell (11Shulga N. James P. Craig E.A. Goldfarb D.S. J. Biol. Chem. 1999; 274: 16501-16507Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). As described we have used proteins containing GFP and distinct of the hsp70 Ssa4p for In cells, the fusion protein GFP-Ssa4p-(16–642), which contains amino of the sequence of Ssa4p amino was both nuclear and A and contrast, this protein in nuclei of stationary phase cells and the nucleus when cells were provided with fresh medium containing glucose and This in the cytoplasm not gene glucose inhibits transcription from the GAL1 promoter. of from the nucleus was of the carbon in the growth medium as the was for cells with such as and nuclear export of was an active process that was inhibited cells were kept at and For we have analyzed the localization of the GFP and GFP the nucleus and cytoplasm under different growth NLS-GFP, a for classical nuclear transport, in nuclei of cells between nucleus and cytoplasm in starving cells and Ref. U., Rassadi, R., and Chiu, J. (September 8, 2000) FASEB J. 10.1096/fj.99-99-0751fje.Google Scholar). Nuclear accumulation of in starving cells was reversible, and transfer to fresh medium its export into the cytoplasm. Nucleocytoplasmic trafficking in particular, nuclear export of several proteins is sensitive to N. H. T. M. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google S. N. A. EMBO J. 16: PubMed Scopus Google in Ref. 8Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar). stationary phase cells were transferred to fresh medium containing both hydrogen peroxide and diethyl maleate inhibited nuclear export of the fusion protein 2, In contrast, the protein synthesis cycloheximide effect on nuclear and in the as was in cells A and and in Fig. To of Ssa4p that are sufficient for nuclear accumulation when cells enter stationary a fusion of the amino of Ssa4p to GFP was This fusion protein, referred to as the bipartite NLS in In cells, was in the cytoplasm and the nucleus A and to GFP-Ssa4p-(16–642). Moreover, in nuclei of starving cells that the N-terminal segment of Ssa4p was sufficient to nuclear accumulation in stationary phase cells. As a for a non-nuclear protein, the distribution of was also This protein was with nuclei and It be that nuclear accumulation of GFP-Ssa4p-(16–642), or Star-β-galactosidase was and a of the protein was in the cytoplasm. Nuclear accumulation was also and in early stationary phase cells. of Ssa4p are The sequence to as Star sequence, for the consensus sequence for hydrophobic nuclear export signals N. H. T. M. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). when fused to we not nuclear not between nucleus and cytoplasm in cells and in nuclei of starving cells and different nuclear accumulation of in cells. In due to its into nuclei and may be when cells are This an with nuclear proteins which that from into the cytoplasm. the Star sequence as an NLS that is active under conditions. a of nuclear and nuclear import may concentrate in nuclei reporter proteins containing the Star sequence. To to these we have the fusion protein This fusion protein is too large to the nuclear by its nuclear localization transport into nuclei by an active As shown in Fig. Star-β-galactosidase was to accumulate in nuclei of stationary phase cells, the that the Star sequence can as an To the distribution of and Star-β-galactosidase under conditions, both proteins were in the cell and in as well as stationary phase cells and Star-β-galactosidase and in nuclei when cells were entering stationary phase Upon of cells for several in the of and Star-β-galactosidase were and we a concentration of the reporter proteins in nuclei not This most protein in starving cells. We have the nuclear accumulation of in yeast strains carrying a deletion in of the β-importin With the shown in Fig. and in we the localization of in cells during logarithmic growth and in stationary phase. All of the between nucleus and cytoplasm when cells were in nuclei when were in early stationary phase. These are with the that the concentration of the protein in nuclei not depend on particular β-importin and may be by nuclear of and Star-β-galactosidase in wild type and mutant wild type and mutant yeast strains for of the β-importin and Star-β-galactosidase were Cells were analyzed during logarithmic growth and when entering stationary as It is shown whether the signal was in nuclei or cytoplasm that reporter proteins were in nucleus and nuclear accumulation was for of the β-importin genes that have been are in in a In wild type and mutant yeast strains for of the β-importin and Star-β-galactosidase were Cells were analyzed during logarithmic growth and when entering stationary as It is shown whether the signal was in nuclei or cytoplasm that reporter proteins were in nucleus and nuclear accumulation was for of the β-importin genes that have been are in To determine whether the deletion of a β-importin gene affects active nuclear import mediated by the Star sequence, we the distribution of Star-β-galactosidase during logarithmic and stationary of the As shown in Fig. 6 and in most of the deletion were to accumulate Star-β-galactosidase in the that they are not required for nuclear import mediated by the Star sequence. cells lacking a gene to concentrate Star-β-galactosidase in nuclei of early stationary phase cells that Nmd5p a role in these transport As a of of Star-β-galactosidase may in of protein, which its nuclear To of gene we the Star-β-galactosidase gene into carrying the promoter D. M. PubMed Scopus Google Scholar). when we Star-β-galactosidase cells with these the reporter protein not concentrate in nuclei when cells stationary phase not These the that Nmd5p is involved in nuclear import mediated by the Star sequence. in cells to in response to various of stress, proteins may alter their In particular, trafficking between nucleus and cytoplasm is in cells to oxidative or osmotic stress (reviewed in Ref. 8Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar). changes in nutrient concentration have been shown to alter protein localization (6DeVit M.J. Johnston M. Curr. Biol. 1999; 9: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (159) Google C. J. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google G. P. E. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). For instance, the distribution of and of protein kinase A in S. cerevisiae depends on the carbon G. P. E. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the distribution of is in response to changes C. J. J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The effect of nutrient depletion on nucleocytoplasmic transport of heat shock proteins, however, has not been Proteins of the hsp70 family are in the stress a process that is We have now analyzed the effect of starvation on Ssa4p localization in To this distinct of Ssa4p were fused to the non-nuclear reporter proteins GFP or and in nuclei of starving cells during early stationary that the N-terminal domain of Ssa4p is sufficient for nuclear is lacking the bipartite NLS in a classical NLS is not required to concentrate the protein in the nuclei of starving cells. the N-terminal domain of Ssa4p we have identified a short of hydrophobic amino termed Star (for starvation), which is sufficient to nuclear concentration in cells. Thus, both as well as Star-β-galactosidase accumulate in nuclei of early stationary phase cells. the sequence of Star is different in both fusion proteins, out the that an nuclear targeting sequence was by the Star sequence to a reporter Proteins that the of the nuclear pore enter the nucleus by active transport, whereas molecules can diffuse nuclear pore We have that Star-β-galactosidase concentrates in nuclei of starving cells, whereas NLS-GFP, a for classical nuclear protein to This in the for nuclear accumulation and the cellular Star-β-galactosidase and classical is by of mutant yeast strains that carry a deletion of of the β-importin we have identified Nmd5p as a β-importin that an essential role in nuclear accumulation of Star-β-galactosidase when cells enter stationary phase. contrast, is to be into nuclei by the classical nuclear import α-importin and the β-importin and this pathway is in starving cells. Moreover, nuclear accumulation in starving cells is not a of proteins carrying a nonclassical For instance, the yeast protein Npl3p contains a nonclassical NLS to concentrate in nuclei of starving cells (7Stochaj, U., Rassadi, R., and Chiu, J. (September 8, 2000) FASEB J. 10.1096/fj.99-99-0751fje.Google Scholar). We that the Star sequence as a nonclassical NLS in starving cells. is a gene that encodes a nuclear carrier which the mitogen-activated protein kinase Hog1p and the transcription factor into nuclei (5Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5506-5614Crossref Scopus (345) Google M. L. G. J. Cell Biol. 1998; PubMed Scopus Google Scholar). We have now identified the Star sequence as a signal that mediates nuclear transport via Nuclear import via Nmd5p by direct of a protein to Nmd5p or by a that a between Nmd5p and the Star sequence. At present, targeting signals recognized by Nmd5p are not and have to determine whether Nmd5p and the Star sequence It be that we not nuclear of Star-β-galactosidase in cells carrying a deletion of This that of Star-β-galactosidase enter the nucleus by an Like may accumulate in nuclei by a nuclear import pathway that is in starving cells Fig. nuclear accumulation not depend on of the β-importins we have cells lacking a gene in nuclei when entering stationary phase. The of these is that nuclear accumulation of is by nuclear to its in and out of the nucleus. the nuclear the Star sequence may to that in nuclei the of for Star-β-galactosidase and we that the Star sequence has two active nuclear a reaction that depends on the Star sequence with nuclear that its from the nucleus, a reaction of signal import and to be when cells enter stationary phase. Nuclear accumulation of is reversible, that starving cells, when provided with fresh export the fusion protein into the cytoplasm. is too large to the nucleus by nuclear export has to be mediated by an active This is in with that nuclear cells are transferred to fresh medium at Moreover, nuclear export of is sensitive to that are to other nuclear export processes N. H. T. M. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). data to the following for protein localization mediated by the Star sequence In cells a protein can be in the nucleus and in the cytoplasm. The protein in nuclei of starving cells, a process that is mediated by nuclear with the Star sequence as a nonclassical nuclear by the Star sequence into the cytoplasm. At present, we can the role of Ssa4p nuclear concentration in early stationary phase. Like nuclear accumulation of hsp70s upon heat shock, concentration of Ssa4p in nuclei may the of proteins in cells. provided with fresh Ssa4p is from nuclear and may in of nuclear Ssa4p nuclear export and localize the protein to both nucleus and cytoplasm as in cells. We V. Doye (Paris), D. Goldfarb (Rochester, and G. Schlenstedt (Hamburg, for with strains and the plasmid We are to A. for of the

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