TWO CRM1-DEPENDENT NUCLEAR EXPORT SIGNALS ARE INVOLVED IN THE REGULATION OF HIF1/SIMA SUBCELLULAR LOCALIZATION.

NURIA ROMERO AND PABLO WAPPNER

Córdoba- Argentina

Workshop; International Workshop- Lastest concepts in developmental biology; 2006

The heterodimeric transcription factor HIF (Hypoxia Inducible Factor) has a central and conserved role in oxygen homeostasis throughout the animal kingdom, ranging from nematode worms to humans. HIF is composed of two basic-helix-loop-helix (bHLH)-PAS protein subunits, HIFa and HIFb. We have identified the Drosophila bHLH-PAS proteins Sima and Tango as the HIF1a and HIF1b homologues respectively. We have previously reported that in normoxia Sima is localized in the cytoplasm and in hypoxia it accumulates in the nuclear compartment. Here we show that removal of 47aa from the N-terminus of the protein was sufficient to promote Sima nuclear accumulation, consistent with the occurrence of two canonical CRM1-dependent Nuclear Export Signal motifs (NES) in this region that are structurally conserved in other proteins of the bHLH-PAS family. One of these NES was able to promote nuclear export of a GFP reporter construct in S2 cells, in a leptomycinB-sensitive manner. Mutagenesis of two-conserved leucine residues in this NES caused abrogation of nuclear export activity, confirming the functionality of this signal. To investigate the role of these two NESs in vivo we over-expressed Sima protein in Drosophila transgenic embryos and studied subcellular localization by fluorescence microscopy.  Deletion of either NESs resulted in the inhibition of nuclear export, indicating that both consensus sequences are functional in vivo in the context of the full-length protein. Using the same experimental setting, we mutagenized the two NESs and found that nuclear export upon re-oxygenation was impaired in both cases. We conclude that the bHLH-PAS protein Sima is exported from the nucleus to the cytoplasm in a mechanism mediated by the action of at least two CRM1-dependent NES localized in the bHLH domain, and that these signals are important for rapid nuclear clearance upon cessation of the hypoxic stimulus.