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.     

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.     

Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein

We previously reported that the nuclear localization signal (NLS) peptides stimulate the in vitro phosphorylation of several proteins, including a 34 kDa protein. In this study, we show that this specific 34 kDa protein is a novel murine leucine-rich acidic nuclear protein (LANP)-like large protein (mLANP-L). mLANP-L was found to have a basic type NLS. The co-injection of Q69LRan-GTP or SV40 T-antigen NLS peptides prevented the nuclear import of mLANP-L. mLANP-L NLS bound preferentially to Rch1 and NPI-1, but not to the Qip1 subfamily of importin alpha. These findings suggest that mLANP-L is transported into the nucleus by Rch1 and/or NPI-1.     

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.     

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.     

The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.     

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 beta mediates nuclear translocation of Smad 3

Smad proteins are intracellular mediators of transforming growth factor-beta (TGF-beta) and related cytokines. Although ligand-induced nuclear translocation of Smad proteins is clearly established, the pathway mediating this import is yet to be determined. We previously identified a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signal transduction. This basic motif (Lys(40-)Lys-Leu-Lys-Lys(44)), conserved among all the pathway-specific Smad proteins, is required for Smad 3 nuclear import in response to ligand. Here we studied the nuclear import pathway of Smad 3 mediated by this NLS. We demonstrate that the isolated Smad 3 MH1 domain displays significant specific binding to importin beta, which is diminished or eliminated by mutations in the NLS. Full-size Smad 3 exhibits weak but specific binding to importin beta, which is enhanced after phosphorylation by the type I TGF-beta receptor. In contrast, no interaction was observed between importin alpha and Smad 3 or its MH1 domain, indicating that nuclear translocation of Smad proteins may occur through direct binding to importin beta. We propose that activation of all of the pathway-specific Smad proteins (Smads 1, 2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then binds directly to importin beta and triggers nuclear translocation.     

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.     

14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1)

p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.     

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.     

Classical nuclear localization signals: definition, function, and interaction with importin alpha

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.