Nuclear localization signal and protein context both mediate importin alpha specificity of nuclear import substrates

The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha 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 alpha3, 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 alpha binding and transport.     

Nucleocytoplasmic protein transport and recycling of Ran

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.     

Importin beta-depending nuclear import pathways: role of the adapter proteins in the docking and releasing steps

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin beta. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin beta is different for each pathway. Although the adapter for cNLS-protein is importin alpha, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin beta results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin beta documented that SPN1 and importin alpha have different binding sites on importin beta. Importin beta fragment 1-618, which binds to SPN1 but not to importin alpha, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin beta-binding domain of the adapters influences the release of the cargo into the nucleoplasm.     

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.     

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.     

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.     

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.     

A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.     

Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.     

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.     

Nuclear import of the U1A splicesome protein is mediated by importin alpha /beta and Ran in living mammalian cells

U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.     

Core histones and linker histones are imported into the nucleus by different pathways

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.     

Characterization of an unusual importin alpha binding motif in the borna disease virus p10 protein that directs nuclear import.

Nuclear import of many cellular and viral proteins is mediated by short nuclear localization signals (NLS) that are recognized by intracellular receptor proteins belonging to the importin/karyopherin alpha and beta families. The primary structure of NLS is not well defined, but most contain at least three basic amino acids and harbor the relative consensus sequence K(K/R)X(K/R). We have studied the nuclear import of the Borna disease virus p10 protein that lacks a canonical oligobasic NLS. It is shown that the p10 protein exhibits all characteristics of an actively transported molecule in digitonin-permeabilized cells. Import activity was found to reside in the 20 N-terminal p10 amino acids that are devoid of an NLS consensus motif. Unexpectedly, p10-dependent import was blocked by a peptide inhibitor of importin alpha-dependent nuclear translocation, and the transport activity of the p10 N-terminal domain was shown to correlate with the ability to bind to importin alpha. These findings suggest that nuclear import of the Borna disease virus p10 protein occurs through a nonconventional karyophilic signal and highlight that the cellular importin alpha NLS receptor proteins can recognize nuclear targeting signals that substantially deviate from the consensus sequence.     

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.     

Phospholipid scramblase 1 contains a nonclassical nuclear localization signal with unique binding site in importin alpha

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin alpha/beta. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS (257)GKISKHWTGI(266). This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 A crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin alpha. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin alpha, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin alpha.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.