Developmental regulation of the heat shock response by nuclear transport factor karyopherin-alpha3.
During early stages of Drosophila development the heat-shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a Drosophila nuclear transport protein karyopherin-alpha3 (dKap-alpha3). Biochemical methods demonstrate that the dKap-alpha3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-alpha3 protein does not associate with NLSs that contain point mutations, which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-alpha3. Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-alpha3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.
Fang
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Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Chen
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T
Tran
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Parker
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CS
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D000076249:Heat Shock Transcription Factors; D018869:Heat-Shock Response; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D028901:alpha Karyopherins; D028961:beta Karyopherins
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D000076249:Heat Shock Transcription Factors; C053134:Hsf protein, Drosophila; D019476:Insect Proteins; C106306:KPNA3 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014157:Transcription Factors; D028901:alpha Karyopherins; D028961:beta Karyopherins