Nuclear import of CREB and AP-1 transcription factors requires importin-beta 1 and Ran but is independent of importin-alpha

Although the specific role of transcription factors (TFs) is nuclear, surpr isingly little is known in quantitative terms regarding the pathways by whi ch TFs localize in the nucleus. In this study, we use direct binding assays , native gel electrophoresis, and fluorescence polarization measurements to show for the first time that the cAMP-response element binding protein (CR EB) and related AP-1 and jun and fos constituents are recognized by importi n beta1 (Imp beta) with nanomolar affinity, We reconstitute the nuclear imp ort of these TFs in vitro, demonstrating dependence on cytosolic factors, a nd show that this is due to the requirement for Imp beta, since antibodies to Imp beta, but not to importin alpha (Imp alpha), inhibit nuclear accumul ation significantly. We show that Imp is necessary and sufficient for docki ng of CREB at the nuclear envelope; that Ran is essential for CREB nuclear import is demonstrated by the reduction of nuclear accumulation effected by RanGTP gammaS but not RanGDP and by dissociation of the Imp beta -CREB-GFP complex by RanGTP gammaS but nor RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Imp beta1- an d Ran-mediated nuclear impart pathway for CREB and related constitutively n uclear TFs, which is Imp alpha -independent and thus distinct from import p athways utilized by inducible TFs.