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PubMed2003

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding.

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.

Alleles↗

PubMed2000

Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3.

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.

Animals↗

PubMed2001

Karyopherins and nuclear import.

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.

Active Transport, Cell Nucleus↗

PubMed1999

Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin alpha2beta1 heterodimers.

L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor-mediated manner into the nuclei of digitonin-permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via karyopherin alpha2beta1 heterodimers. Anti-karyopherin-beta1 and anti-karyopherin alpha2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using karyopherin alpha2, beta1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to karyopherin alpha2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the karyopherin alpha2beta1 pathway.

Amino Acid Sequence↗

PubMed2002

Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal.

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.

Binding Sites↗

PubMed1997

Karyopherin beta2 mediates nuclear import of a mRNA binding protein.

We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 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 beta1, karyopherin beta2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both beta1 and beta2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, beta2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of beta2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of beta2 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 beta2 inhibited karyopherin alpha/beta1-mediated import of a classical NLS containing substrate and, vice versa, beta1 inhibited beta2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta1 and beta2 to repeat nucleoporins.

Amino Acid Sequence↗

PubMed2004

Karyopherins: from nuclear-transport mediators to nuclear-function regulators.

The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.

Active Transport, Cell Nucleus↗

PubMed2001

Genomic structure of karyopherin alpha2 ( KPNA2) within a low-copy repeat on chromosome 17q23-q24 and mutation analysis in patients with Russell-Silver syndrome.

Human karyopherin alpha2 (KPNA2), a member of the karyopherin alpha family, plays a key role in the nuclear import of proteins with a classical nuclear localization signal (NLS). KPNA2, as part of a karyopherin alpha-beta heterodimer, directly binds to the NLS of proteins and functions as an adaptor that binds NLS-containing proteins via karyopherin beta to the nuclear pore complex. The NLS protein-receptor complex is translocated through the pore by an energy-dependent mechanism. Recently, we have identified and mapped the gene for KPNA2 in close proximity to a translocation breakpoint within 17q23-q24 associated with Russell-Silver syndrome (RSS). Therefore, we considered KPNA2 as a positional candidate gene for this heterogeneous disorder. RSS is mainly characterized by pre- and postnatal growth retardation, lateral asymmetry, and other dysmorphic features. Here, we present the genomic organization of the human KPNA2 gene with 11 exons spanning approximately 10 kb on chromosome 17q23-q24. Screening for mutations within all exons and adjacent intronic sequences from 31 unrelated RSS patients revealed three single nucleotide polymorphisms (SNPs) in exons 1, 5, and 7, and five SNPs in introns 1, 4 (2 SNPs), 8, and 9, respectively. No disease-related mutation was identified by comparing the sequence data of the RSS patients with their clinically normal parents and controls.

Abnormalities, Multiple↗

PubMed2003

An intracellular targeted NLS peptide inhibitor of karyopherin alpha:NF-kappa B interactions.

The nuclear import of transcription factors involves proteins termed karyopherins. Previously, we described an intracellular targeted dual nuclear localization sequence (NLS) peptide inhibitor of processes dependent upon the transcription factor NF-kappa B. We have now developed a homogeneous solution based assay and show that NF-kappa B interacts with karyopherin alpha and that the dual NLS peptide inhibits this interaction. We also show that both L- and D-amino acid containing peptides bind well to karyopherin alpha 2, whereas, the L-amino acid peptides bind more efficiently than the D-amino acid peptide to karyopherin alpha1.

Animals↗

PubMed1997

Differential expression and sequence-specific interaction of karyopherin alpha with nuclear localization sequences.

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins alpha, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1alpha are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins alpha to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.

Amino Acid Sequence↗

PubMed2000

The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin alpha3/Qip.

RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.

Amino Acid Sequence↗

PubMed2002

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes.

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.

Binding Sites↗

PubMed2006

Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1 nuclear accumulation.

Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-alpha/beta) and the ability of cells to respond to IFN-alpha/beta or IFN-gamma. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-alpha/beta production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-alpha/beta and IFN-gamma signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-alpha/beta and IFN-gamma signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin alpha1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin alpha2, alpha3, or alpha4. Overexpression of VP24 results in a loss of karyopherin alpha1-PY-STAT1 interaction, indicating that the VP24-karyopherin alpha1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.

Animals↗

PubMed2004

The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions.

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.

Amino Acid Sequence↗

PubMed2004

Characterization of karyopherin cargoes reveals unique mechanisms of Kap121p-mediated nuclear import.

In yeast there are at least 14 members of the beta-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast beta-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.

Amino Acid Sequence↗

PubMed1997

Isolation and mapping of karyopherin alpha 3 (KPNA3), a human gene that is highly homologous to genes encoding Xenopus importin, yeast SRP1 and human RCH1.

From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human. The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids. The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively. The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system. Eight repeats of the arm motif were well conserved among these proteins. The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic. A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting. The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed1998

Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha.

Selective nuclear import is mediated by nuclear localization signals (NLSs) and cognate transport factors known as karyopherins or importins. Karyopherin alpha recognizes "classical" monopartite and bipartite NLSs. We report the crystal structure of a 50 kDa fragment of the 60 kDa yeast karyopherin alpha, in the absence and presence of a monopartite NLS peptide at 2.2 A and 2.8 A resolution, respectively. The structure shows a tandem array of ten armadillo repeats, organized in a right-handed superhelix of helices. Binding of the NLS peptide occurs at two sites within a helical surface groove that is lined by conserved residues. The structure reveals the determinants of NLS specificity and suggests a model for the recognition of bipartite NLSs.

Amino Acid Sequence↗

PubMed2000

Structure and expression of Xenopus karyopherin-beta3: definition of a novel synexpression group related to ribosome biogenesis.

Karyopherin-beta3 is a nuclear transport receptor that appears to be involved in nuclear import of ribosomal proteins. Here we report on sequence and expression of karyopherin-beta3 in Xenopus. The differential distribution of karyopherin-beta3 mRNA during Xenopus embryogenesis is similar to that of several other protein import factors and of ribosomal proteins. These genes thus define a novel synexpression group in the context of ribosome biogenesis.

Amino Acid Sequence↗