Although only 44% identical to human karyopherin a,, human karyopherin a2 (Rchl protein) substituted for human karyopherin a, ( (3, 4). Indeed, mapping of the docking site(s) for Nup98 showed it (them) to be located in the repeat-containing N-terminal half of Nup98 (12). Finally, the repeat-containing nucleoporins also contain binding sites for plO (M. Matunis, G.B., and M.H., unpublished data). It has been proposed that the repeatcontaining nucleoporins serve as a stationary phase and the transport factors as the mobile phase in transport across the NPC (12).We showed previously that the a subunit serves as an NLS receptor and proposed that the X3 subunit serves as an adaptor that binds to the a subunit-NLS substrate complex and to the repeat-containing nucleoporins (4). Here we show that the previously used karyopherin a subunit [corresponding to hSRP-1/NPI-1 (14, 15)], now termed karyopherin a1, can be replaced by karyopherin a2 [corresponding to the hSRP-1-related Rchl (16)]. Although karyopherin a, and a2 are only 44% identical, we did not detect any functional difference. We also show that karyopherin 3 is localized in the cytoplasm and at the nuclear rim and is largely lost after digitonin permeabilization of cells. Unlike karyopherin a, which alone cannot bind to the nuclear envelope of digitonin-permeabilized cells, karyopherin , can bind without karyopherin a being present. In an import reaction with all recombinant transport factors, the karyopherin a subunits, Ran, and plO are shown to enter the nucleus, whereas the ,B subunit remains at the nuclear envelope. This result suggests that the heterodimeric karyopherin complex is dissociated during transport. Overlay binding assays showed that the ,B subunit binds to the repeatcontaining nucleoporins. These data are consistent with the previously proposed function of karyopherin J3 as an adaptor between karyopherin a/NLS substrate complex and repeatcontaining nucleoporins.Abbreviations: HSA, human serum albumin; NLS, nuclear localization sequence; NPC, nuclear pore complex; FITC, fluorescein isothiocyanate.

The intracellular pathway of human angiogenin in calf pulmonary artery endothelial (CPAE) cells has been studied by immunofuorescence microscopy. Proliferating CPAE cells specifically endocytose native angiogenin and translocate it to the nucleus, where it accumulates in the nudeoli. Nuclear translocation of angiogenin does not occur in nonproliferative, confluent CPAE cells. These cells were previously found to express an angiogenin-binding protein (AngBP) that was identified as smooth muscle a-actin. Exogenous actin, an anti-actin antibody, heparin, and heparinase treatment all inhibit the internalization of angiogenin, suggesting the involvement of cell surface AngBP/actin and heparan sulfate proteoglycans in this process. It has been established that two regions of angiogenin are essential for its angiogenic activity, one is its endothelial cell binding site and the other its catalytic site capable of cleaving RNA. CPAE cells do not internalize four enzymatically active angiogenin derivatives whose cell binding site is modified, but they do internalize two enzymatically inactive mutants whose cell binding site is intact. Thus, the putative cell binding site of angiogenin is necessary for both endocytosis and nuclear translocation, but the catalytic site is not. Three other angiogenic molecules are also translocated to the nucleus of growing CPAE cells. Overall, the results suggest that nuclear translocation of angiogenin and other angiogenic molecules is a critical step in the process of angiogenesis.

We have fluorescently labeled one of the eight genomic segments of influenza virus RNA and a recombinant influenza viral protein, the nucleoprotein (NP), to investigate the requirement for their uptake into nuclei of digitonin-permeabilized cells. We found that the influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein. Thus, at 0°C it docks at the nuclear envelope only in the presence of the heterodimeric karyopherin (either karyopherin ␣1␤ or karyopherin ␣2␤), and docking is competitively inhibited by an unlabeled NLS containing substrate. Like other NLS-containing proteins, at 20°C NP is imported into the nucleus after further addition of the GTPase Ran and of p10. In contrast, the fluorescently labeled, 890-nucleotide-long viral RNA segment does not dock to the nuclear envelope or enter the nucleus either in the presence of exogenous cytosol or of karyopherin heterodimer, Ran, and p10. However, in the presence of NP the RNA is able to dock and enter the nucleus with transport requirements indistinguishable from those for docking and entry of NP. These data indicate that uptake of the influenza virus RNA segment is not via a signal in the RNA but via an NLS of a viral protein such as NP.

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitoninpermeabilized cells and also had molecularly cloned and sequenced its 97-kDa 13 subunit. We now report that the karyopherin a subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin a or .8 subunit, we showed that neither karyopherin a nor karyopherin 1 alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin a and .8 subunits and import substrate as a ligand, we found that only karyopherin a bound ligand. We suggest that karyopherin 1 functions as an adaptor that binds both to karyopherin a and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin a-substrate across the NPC.In an in vitro nuclear import system that utilizes a nuclear localization sequence (NLS)-containing import substrate and digitonin-permeabilized cells (retaining intact importcompetent nuclei but being largely depleted of cytosolic proteins), import is dependent on exogenous cytosol (1, 2). The required cytosolic factors appear to be highly conserved because an amphibian (Xenopus ovary) cytosol can compensate for leaked out cytosol from digitonin-permeabilized mammalian cells (2). Biochemical subfractionation of the Xenopus ovary cytosol yielded two fractions (A and B) with distinct activities (2). Fraction A functions in the recognition of the NLS-containing substrate and its docking at the nuclear envelope, whereas fraction B is required for subsequent nuclear internalization of the docked substrate (2). The active components of both Xenopus cytosolic subfractions have been purified. The small GTP-binding protein designated "Ran" (3) for ras-related nuclear protein and a Ran-interactive protein, plO (4), represent fraction B's activity, whereas a 9-S complex of three proteins of 54, 56, and 97 kDa represents fraction A's activity (5). Because of its function in docking the import substrate prior to nuclear internalization, the 9-S complex has been named karyopherin, with the 54-and 56-kDa proteins termed its a subunit and the 97-kDa protein named its 13 subunit (5). Using bovine erythrocyte cytosol and based on crosslinking by an NLS-containing peptide, two proteins of 54 and 56 kDa were purified and termed the NLS receptor(s) (7). Subsequent purification of a third bovine erythrocyte protein of 97 kDa (p97) showed that both p97 and p54/56 were required for docking...

We have cloned and sequenced cDNA for human karyopherin ␤2, also known as transportin. In a solution binding assay, recombinant ␤2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin ␤1, karyopherin ␤2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both ␤1 and ␤2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, ␤2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of ␤2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of ␤2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin ␤2 inhibited karyopherin ␣͞␤1-mediated import of a classical NLS containing substrate and, vice versa, ␤1 inhibited ␤2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of ␤1 and ␤2 to repeat nucleoporins.Import of proteins containing a nuclear localization sequence (NLS) into digitonin-permeabilized cells is mediated by soluble transport factors. A heterodimer, termed karyopherin or importin, recognizes the NLS protein in the cytoplasm via its ␣ subunit and, via its ␤ subunit, docks the complex to a subset of peptide repeat containing nucleoporins (1-10). The GTPase Ran (11, 12) and a Ran interacting protein, termed p10 (or NTF2) (13, 14), then mediates release and GTP-hydrolysisdependent transport of the NLS protein and karyopherin ␣ into the nucleus with karyopherin ␤ staying behind at the nuclear pore complex (10,(15)(16)(17). Homologs of these transport factors also have been identified in yeast and recombinant Kap60p͞Srp1p (karyopherin ␣) and Kap95p (karyopherin ␤) can substitute for their mammalian homologs in docking NLS protein to the nuclear rim of digitonin-permeabilized mammalian cells (18).Studies in yeast have revealed the existence so far of three proteins that are both structurally and functionally related to Kap95p (ref. 19; M. P. Rout, G.B., and J. D. Aitchison, unpublished data) and therefore have been classified as members of the yeast ␤ karyopherin family. All four yeast ␤ karyopherins [Kap95p, Kap104p, Pse1p (20), and Kap123p]

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The a subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the 13 subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p1O, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin 3's binding sites both for Ran-GTP and for karyopherin a. We discovered that karyopherin .3's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin a bind to overlapping sites on karyopherin 18. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin ca and binds to karyopherin 13. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin 13 to a cluster of basic residues analogous to those previously shown to constitute karyopherin ca's binding site to karyopherin 1.

The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline et al., 2003). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence (76IRTLEDLLM84) changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.