Developmental Regulation of the Heat Shock Response by Nuclear Transport Factor Karyopherin-a3

Xiang-dong Fang¹, Tianxin Chen¹, Kim Tran and Carl S. Parker^1,2

¹Division of Chemistry and Chemical Engineering

California Institute of Technology

Pasadena, California

91125

Abstract

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 nuclear transport protein Drosophila karyopherin-a3 (dKap-a3).  Biochemical methods demonstrate that the dKap-a3 protein binds tightly to the NLS.  Furthermore the dKap-a3 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-a3.  Previous studies from other laboratories have demonstrated 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-a3 protein until 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.