Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.

The "classical" nuclear import pathway depends on importins ␣ and ␤. Humans have only one importin ␤, while six ␣ importins have been described. Whether or not distinct ␣ importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human ␣ importins in HeLa cells. Down-regulation of importins ␣3, ␣5, ␣7, and ␤ strongly inhibited HeLa cell proliferation, while down-regulation of importins ␣1 and ␣4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any ␣ importin, indicating that the importin ␣/␤ pathway was generally not affected. In contrast, importin ␣3 or ␣5 downregulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant ␣ importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin ␣3 in both cases. Thus, importin ␣3 is the only ␣ importin responsible for the classical nuclear import of RCC1 in living cells.Macromolecule transport between cell cytosol and nucleus takes place through the nuclear pore complexes (NPC) (2, 41). Import substrates possess nuclear localization signals (NLS), required for recognition by distinct nuclear import factors. The so-called classical nucleocytoplasmic import pathway is mediated by the importin ␣/␤ heterodimer, also known as karyopherin ␣/␤ (for reviews, see references 23, 34, and 43). Importin ␣ acts as an adapter by binding both the import substrate and importin ␤. The trimeric import complex docks to the NPC via importin ␤ and translocates into the nucleus. Recently, Npap60/Nup50 was identified as an additional mammalian cofactor for importin ␣/␤-dependent nuclear protein import (22). While only one importin ␤ isoform exists, six human ␣ importins have been described (3,4,18,21,28,36,42). In contrast, the yeast Saccharomyces cerevisiae possesses only one gene for importin ␣, which is essential (45).The ␣ importins are grouped into three subfamilies based on sequence homology. The first subfamily consists of importin ␣1/Rch1. Its most closely related homologue, importin ␣2, has been found in Xenopus laevis and other vertebrates but not in mammals. Importins ␣3/Qip1 and ␣4/hSRP1␥ are members of the second subfamily. The third subfamily consists of importins ␣5/hSRP1, ␣6, and ␣7 (18). The isoforms of one subfamily are highly homologous, showing about 85% sequence identity and differing mostly in regions outside the NLS binding pockets (18,21,26). Although the ␣ importins differ in their cell-and tissue-specific expression patterns, most are expressed ubiquitously (16,18,20,21,28,31,40). Only importin ␣6 expression seems to be restricted to the testis (21). The reason for the importin ␣ diversity in higher eukaryotes, especially in humans and other vertebrates, is unknown. Invertebrates such as Caenorhabditis elegans and Drosophila melan...

The "classical" nuclear protein import pathway depends on importin ␣ and importin ␤. Importin ␣ binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin ␤-dependent import pathway. In humans, only one importin ␤ is known to interact with importin ␣, while six ␣ importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular ␣ importins. Whether the NLS is sufficient to mediate importin ␣ specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin ␣3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin ␣ binding and transport.Nuclear import substrates possess nuclear localization signals (NLSs) required for recognition by distinct nuclear import factors. The so-called "classical" NLS consists of either one cluster of basic amino acids (monopartite NLS) or two clusters of basic amino acids separated by a linker (bipartite NLS). Nuclear transport of substrates bearing a classical NLS is mediated by the importin ␣/␤ heterodimer, also known as karyopherin ␣/␤. Importin ␣ functions as an adapter by binding both the import substrate via the NLS and importin ␤. Importin ␤ docks the ternary import complex at the nuclear pore complex and facilitates its translocation through the nuclear pore complex into the nucleus (13,20). In addition, importin ␣ and ␤ are also involved in other processes associated with nuclear functions, ranging from spindle formation to nuclear envelope assembly (17-19, 35, 48, 52).Importin ␣ is composed of a short basic N-terminal importin ␤ binding domain (14, 50) and a large NLS binding domain comprised of 10 tandem armadillo (ARM) repeats (4, 24). The series of ARM repeats generates a superhelical structure that has a shallow, concave NLS binding groove containing two NLS binding sites consisting of ARM repeats 1 to 4 (major site) and 4 to 8 (minor site) (4, 11). Bipartite NLS sequences span the two binding sites, with each site recognizing one of the basic clusters (3, 10, 11). Monopartite NLS sequences are able to bind both sites, but only the binding at the major site, corresponding to the C-terminal basic cluster of the bipartite NLS, is likely to be physiologically relevant (3,4,11,12). The N-terminal importin ␤ binding domain of importin ␣ serves a dual role. It binds to importin ␤ but also contains an autoinhibitory sequence that mimics an NLS. This autoinhibitory sequence interacts with the NLS binding domain when importin ␣ is not bound to importin ␤ and/or to an NLS cargo (9,24,34). Accordingly, the affinity of importin ␣ for import substrates is increased in the presence of importin ␤ (2, 9, 41).While only one importin ␤ isoform exists for interaction with impo...