Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.     

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import [published erratum appears in J Cell Biol 1992 Jul;118(1):215]

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol. NBPs are phosphorylated and phosphatase treatment abolished NLS binding. The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells. Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP. In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope. Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus. Anti-NBP antibodies specifically inhibited the binding part of this import reaction. These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.     

RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha3

The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-alpha, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-alpha family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-alpha3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase-green fluorescent protein-RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-alpha or importin-beta or by the absence of importin-alpha. Binding assays using recombinant importin-alpha1, -alpha3, -alpha4, -alpha5, and -alpha7 revealed a preferential interaction of the RanBP3 NLS with importin-alpha3 and -alpha4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-alpha3. These results demonstrate that members of the importin-alpha family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.     

Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-alpha, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-alpha. The nucleoplasmin NLS-importin-alpha complex shows, for the first time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein.     

Cytosolic import factor- and Ran-independent nuclear transport of ribosomal protein L5

Ribosomal protein L5 is a shuttling protein that, in Xenopus oocytes, is involved in the nucleocytoplasmic transport of 5S rRNA. As demonstrated earlier, L5 contains three independent nuclear import signals (NLSs), which function in oocytes as well as in somatic cells. Upon physical separation, these NLSs differ in respect to their capacity to bind to nuclear import factors in vitro and to mediate the nuclear import of a heterologous RNP in vivo. As reported in this communication, analysis of the in vitro nuclear import activity of these three NLSs reveals that they also differ in respect to their requirements for cytosolic import factors and Ran. Nuclear import mediated by the N-terminal and the central NLS depends on cytosolic import factor(s) and Ran, whereas import via the C-terminal NLS occurs independently from these factors. Thus, the presence of multiple NLSs in ribosomal protein L5 appears to allow for efficient nuclear transport via utilisation of multiple, mechanistically different import pathways.     

Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.

We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2- to 3-fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro.     

In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.     

Kinetic properties of nuclear transport conferred by the retinoblastoma (Rb) NLS

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein central to control of cellular proliferation. We have previously shown that human RB possesses an evolutionarily conserved bipartite nuclear localization sequence (NLS) (KRSAEGSNPPKPLKKLR877) resembling that of nucleoplasmin. Here we analyze the kinetic properties of the RB NLS in detail with respect to recognition by cellular nuclear import factors, the importins (IMPs), and nuclear transport properties, comparing results to those for the NLSs from SV40 large tumor antigen (T-ag) and the Xenopus laevis phosphoprotein N1N2. Binding affinities of different IMPalpha subunits for the Rb NLS, in the absence or presence of IMPbeta subunits were determined, and NLS-dependent nuclear import reconstituted in vitro for the first time using purified IMPalpha/beta subunits together with recombinant human RanGDP and nuclear transport factor 2 (NTF2). RB NLS-mediated transport had a strict requirement for all components, with high NTF2 concentrations inhibiting transport. As in the case of transport mediated by the T-ag- and N1N2-NLSs, nuclear import of an RB-NLS containing beta-Gal fusion protein was reduced or abolished when anti-IMPalpha or beta antibody was added to cytosolic extract, respectively, confirming that RB NLS-mediated nuclear import occurs through action of IMPalpha/beta. We conclude that although mediated by IMPalpha/beta, and similar in most respects to transport mediated by the similarly bipartite N1N2 NLS, nuclear import conferred by the RB NLS has distinct properties, in part due to the affinity of its interaction with IMPalpha.     

Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling

Nuclear import of proteins containing classical nuclear localization signals (NLS) is mediated by the importin-alpha:beta complex that binds cargo in the cytoplasm and facilitates its passage through nuclear pores, after which nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through nuclear pores. However, we show here that the Nup50 N-terminal domain actively displaces NLSs from importin-alpha, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin-alpha:Nup50 complex shows that Nup50 binds at two sites on importin-alpha. One site overlaps the secondary NLS-binding site, whereas the second extends along the importin-alpha C-terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin-alpha export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of nuclear protein import.     

Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.     

Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import

The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-alpha:beta-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-alpha. Residues 1-51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo. We have determined the 2.6 A resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p. Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them. Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p. Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-beta binding (IBB) domain of Kap60p. These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.     

Crystallization and preliminary X-ray diffraction analysis of importin-alpha complexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). The study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-alpha was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 A resolution. Preliminary electron density calculations show that the ligands are present in the crystals.     

Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.     

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.     

Reconstitution of nuclear protein transport with semi-intact yeast cells

We have developed an in vitro nuclear protein import reaction from semi- intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS- containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.     

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.