A Nuclear Export Signal Prevents Saccharomyces cerevisiae Hsp70 Ssb1p from Stimulating Nuclear Localization Signal-directed Nuclear Transport*

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSBgene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei ofxpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Δ cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport innup188-Δ cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.

[1]  E. Craig,et al.  Functional Specificity Among Hsp70 Molecular Chaperones , 1997, Science.

[2]  Ohba Masayuki.  A 70‐kDa heat shock cognate protein suppresses the defects caused by a proteasome mutation in Saccharomyces cerevisiae , 1994 .

[3]  D. DeFranco,et al.  Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen , 1994, Molecular and cellular biology.

[4]  J. Brodsky,et al.  Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum , 1998, FEBS letters.

[5]  Y. Yoneda,et al.  A protein recognized by antibodies to Asp-Asp-Asp-Glu-Asp shows specific binding activity to heterogeneous nuclear transport signals. , 1990, The Journal of biological chemistry.

[6]  R. Mandell,et al.  Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope , 1990, The Journal of cell biology.

[7]  Richard I. Morimoto,et al.  1 Progress and Perspectives on the Biology of Heat Shock Proteins and Molecular Chaperones , 1994 .

[8]  Y. Shi,et al.  The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate , 1992, Molecular and cellular biology.

[9]  J. M. Velazquez,et al.  hsp70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery , 1984, Cell.

[10]  A. Vojtek,et al.  Ras-Raf interaction: two-hybrid analysis. , 1995, Methods in enzymology.

[11]  Craig M. Ogata,et al.  Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK , 1996, Science.

[12]  B. Cullen,et al.  Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay , 1996, Molecular and cellular biology.

[13]  J. Halladay,et al.  Complex multigene family of functionally distinct Hsp70s of yeast. , 1995, Cold Spring Harbor symposia on quantitative biology.

[14]  K. Flaherty,et al.  Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein , 1990, Nature.

[15]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[16]  M Ohba,et al.  Modulation of intracellular protein degradation by SSB1–SIS1 chaperon system in yeast S. cerevisiae , 1997, FEBS letters.

[17]  Y. Yoneda.  How proteins are transported from cytoplasm to the nucleus. , 1997, Journal of biochemistry.

[18]  M. Uhlén,et al.  A synthetic IgG-binding domain based on staphylococcal protein A. , 1987, Protein engineering.

[19]  D. Jeoung,et al.  Human major HSP70 protein complements the localization and functional defects of cytoplasmic mutant SV40 T antigen in Swiss 3T3 mouse fibroblast cells. , 1991, Genes & development.

[20]  A. Dayton,et al.  Tolerance of diverse amino acid substitutions at conserved positions in the nuclear export signal (NES) of HIV-1 Rev. , 1998, Biochemical and biophysical research communications.

[21]  M. Wiedmann,et al.  The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome–nascent chain complex , 1998, The EMBO journal.

[22]  M. Rout,et al.  Karyopherins and kissing cousins. , 1998, Trends in cell biology.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  E. Craig,et al.  Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures , 1985, Molecular and cellular biology.

[25]  F. Melchior,et al.  Mechanisms of nuclear protein import. , 1995, Current opinion in cell biology.

[26]  D. Goldfarb,et al.  In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation , 1996, The Journal of cell biology.

[27]  R. Morimoto,et al.  Molecular chaperones as HSF1-specific transcriptional repressors. , 1998, Genes & development.

[28]  Karsten Weis,et al.  Exportin 1 (Crm1p) Is an Essential Nuclear Export Factor , 1997, Cell.

[29]  J. Hegemann,et al.  Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast , 1996, Yeast.

[30]  M. Miyagi,et al.  Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins , 1992, The Journal of cell biology.

[31]  G. Dreyfuss,et al.  A Novel Receptor-Mediated Nuclear Protein Import Pathway , 1996, Cell.

[32]  R. Morimoto,et al.  Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway , 1998, Molecular and Cellular Biology.

[33]  H. Pelham.  Hsp70 accelerates the recovery of nucleolar morphology after heat shock. , 1984, The EMBO journal.

[34]  Ellson Y. Chen,et al.  Overview of manual and automated DNA sequencing by the dideoxy chain termination method , 1991 .

[35]  D. Görlich,et al.  Nuclear protein import. , 1997, Current opinion in cell biology.

[36]  Michael R. Green,et al.  HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs , 1996, Current Biology.

[37]  D. Goldfarb,et al.  In vivo nuclear transport kinetics in Saccharomyces cerevisiae. , 1998, Methods in cell biology.

[38]  G. Blobel,et al.  The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex , 1996, The Journal of cell biology.

[39]  I. Takenaka,et al.  Hsc70-binding Peptides Selected from a Phage Display Peptide Library that Resemble Organellar Targeting Sequences (*) , 1995, The Journal of Biological Chemistry.

[40]  N. Imamoto,et al.  70-kDa heat-shock cognate protein colocalizes with karyophilic proteins into the nucleus during their transport in vitro. , 1993, Experimental cell research.

[41]  R. Morimoto,et al.  Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding , 1989, The Journal of cell biology.

[42]  A. Rivett.  Intracellular distribution of proteasomes. , 1998, Current opinion in immunology.

[43]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.

[44]  E. Craig,et al.  A heat shock transcription factor with reduced activity suppresses a yeast HSP70 mutant , 1995, Molecular and cellular biology.

[45]  The effect of carboxyl-terminal deletions on the nuclear transport rate of rat hsc70. , 1992, Experimental cell research.

[46]  C. Feldherr,et al.  Evidence for the existence of a novel mechanism for the nuclear import of Hsc70. , 1996, Experimental cell research.

[47]  M. Werner-Washburne,et al.  The translation machinery and 70 kd heat shock protein cooperate in protein synthesis , 1992, Cell.

[48]  E. Craig,et al.  Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.