Hypoxia-inducible transcription factor 1 (HIF-1) mediates the cellular response to hypoxia. HIF-1 activity is controlled via the synthesis, degradation or intracellular localization of its alpha subunit. HIF-1 alpha contains a C-terminal bipartite basic NLS that interacts with importins alpha. We have recently shown that HIF-1 alpha also contains an atypical hydrophobic CRM1- and phosphorylation-dependent NES and can therefore shuttle in and out of the nucleus. We now report that C-terminal NLS mutants of HIF-1 alpha can still enter the nucleus when CRM1-dependent nuclear export is inhibited, indicating that HIF-1 alpha contains an additional functional nuclear import signal. Using an in vitro nuclear import assay, we further show that importins 4 and 7 accomplish nuclear import of HIF-1 alpha more efficiently than the classical importin alpha/beta NLS receptor. Binding assays confirmed the specific physical interaction between HIF-1 alpha and importins 4 and 7. Moreover, the interaction of importin 7 with HIF-1 alpha is mapped at its N-terminal part encompassing the bHLH-PAS(A) domain. By expressing functional HIF-1 in yeast, we show that Nmd5, the yeast orthologue of importin 7, is required for HIF-1 alpha nuclear accumulation and activity. Taken together, our data show that shuttling of HIF-1 alpha. between cytoplasm and nucleus is a complex process involving several members of the nuclear transport receptor family. (C) 2009 Elsevier Inc. All rights reserved.