BACKGROUND
eukaryotic cells are defined by the separation of dna from the rest of the cell by the nuclear envelope, a double bilayer made selectively permeable by nuclear pore complexes   <cit> .
transport of proteins between the nucleus and the cytoplasm is carried out by karyopherins, a family of proteins made up of importins and exportins  <cit> .
classical nuclear localization signal  dependent nuclear import is carried out by importin α and importin β importin α family members bind nls cargo, and bind to importin β through an n-terminal importin β binding domain .
importin β mediates translocation of the nls-importin α-importin β import complex into the nucleus through direct interactions with the npc.
once in the nucleus, rangtp binds to importin β and induces dissociation of the import complex  <cit> .
exportin mediated nuclear export is regulated by rangtp through a related mechanism.
whereas rangtp dissociates import complexes by binding importins, exportins must bind to rangtp in order to bind nuclear export signal  containing cargoes  <cit> .
the heterotrimeric export complex then translocates through the npc and is dissociated in the cytoplasm by rangap stimulated conversion of rangtp to rangdp.
while there are at least  <dig> importin β family members which can bind directly to cargo and mediate import  <cit> , importin β is unique in its ability to bind the importin α family of nuclear transport receptors   <cit> .
importin α binds to two major classes of nls, both characterized by basic amino acids; a monopartite nls, such as the sv <dig> nls, which consists of a single cluster of basic amino acids; and a bipartite nls, such as the retinoblastoma  nls, which consists of two clusters of basic amino acids, separated by a ~ <dig> residue spacer  <cit> .
the architecture of importin α proteins is composed of armadillo  repeats, a three a-helix motif named for the d. melanogaster homologue of β catenin  <cit> .
the binding site for a monopartite nls is located between the 2nd and 4th arm repeats and is called the major site  <cit> .
importin α binds to the c-terminus of bipartite nls sequences with the major site and to the n-terminal element of the bipartite nls using a smaller site created by the 7th and 8th arm repeats called the minor site  <cit> .
the accessibility of these nls binding sites is regulated by an autoinhibitory mechanism.
the ibb of importin α contains basic amino acids that bind to the nls binding surface when the receptor is in an autoinhibited state  <cit> .
importin α binding to nls cargo and to importin β is, therefore, a cooperative process because importin β binding to the ibb relieves the autoinhibition of importin α. relief of autoinhibition facilitates importin α binding to nls cargo.
after nuclear import, the complex is dissociated by the cooperative effects of rangtp binding to importin β, and binding of importin α to cas  <cit> .
cas is an exportin which forms a trimeric complex consisting of cas, rangtp and importin α, and is responsible for recycling importin α to the cytoplasm  <cit> .
yeasts encode a single importin α, but higher eukaryotes encode three importin α subfamilies, designated importin α <dig>  α <dig>  and α <dig>  there are six previously described human importin α forms, each encoded by different gene.
importin α family members show preferences for specific types of nls cargo  <cit> , although there is also some functional redundancy.
most nlss are very similar in sequence leading to the suggestion that importin α specificity is conferred by the protein context of the nls imparted by the cargo  <cit> .
it appears that additional importin α proteins are required for a multicellular organism because different importin α's have specific cargoes to import during differentiation  <cit> .
studies in d. melanogaster have shown that importin α <dig> is not required in adult flies, but is required for oogenesis  <cit>  and that importin α <dig> is favored for a step in larval development  <cit> .
similarly, it has been shown in c. elegans that while importin α <dig> is ubiquitously expressed, α <dig> and α <dig> are expressed only in the germline, and are not sufficient to replace α <dig> in somatic cells  <cit> .
in mouse embryonic stem cells, switching from importin α <dig>  expression to importin α <dig>  expression has been shown to induce neural differentiation  <cit> .
these data suggest that cargo interactions with specific importin α isotypes are important for controlling differentiation pathways.
here we describe a novel importin α in humans, the seventh member of the importin α family, which we term karyopherin alpha  <dig> .
this new α family member is most similar to kpna <dig> and a member of the α <dig> subfamily.
we show that kpna <dig> binds to importin β through an n-terminal ibb, but that its importin β binding affinity appears to be much higher than that of the other kpnas.
kpna <dig> localization is predominantly nuclear in hela cells, highlighting another difference between  <dig> and the other importin α family members.
RESULTS
kpna <dig> is a novel member of the importin alpha family
there are six known members of the human importin α family.
we used blast to query the human genome with the importin α family member kpna <dig>  and retrieved a sequence predicted to encode a seventh member of the importin α family.
because the sequence of the putative importin α was generated by gene prediction, we set out to experimentally validate the expression and function of the predicted importin α gene  as an importin α. using rt-pcr, we amplified a product of the expected size from rna prepared from the prostate cancer cell line lncap.
we sequenced the cdna , and found that it matched that of the predicted gene, leading us to conclude that the gene is transcribed in human cells.
this gene is the seventh member of the importin α family of transport receptors, therefore we named it karyopherin alpha  <dig> .
the complete human importin α family  is shown in table  <dig> with their previous designations, as well as accession and gene id numbers.
alignment of the kpna <dig> protein sequence with kpna's 1- <dig> using clustalw  reveals that kpna <dig> is  <dig> % identical to its closest importin α family member, the prototypical kpna <dig> .
the maximum identity between kpna <dig> and members of the α <dig> and α <dig> subfamilies is only  <dig> % .
kpna <dig>  kpna <dig>  and kpna <dig> share a minimum of  <dig> % identity, while kpna <dig> and kpna <dig> are  <dig> % identical, emphasizing that kpna <dig> is highly divergent from other importin α family members.
the multiple alignment  shows, however, that kpna <dig> is related to other importin α family members throughout the length of the protein, suggesting that it is an nls binding protein.
the number shown is the percent identity between the respective kpna proteins.
here we show the kpna numbering system and the corresponding importin alpha numbers, as well as unique identifiers: accession number, gene id number, and the chromosomal locus.
kpna <dig> homologues are found in vertebrates including mouse , xenopus  and zebra fish .
in all animals, importin α proteins are classified into  <dig> subfamilies, α <dig>  α <dig>  and α <dig>  <cit> .
while vertebrates have seven importin α family members, invertebrates such as drosophila have only  <dig> importin α proteins, one for each subfamily.
we created a phylogenetic tree of the complete human importin α family using protdist .
the drosophila melanogaster importin α proteins were included in the phylogram  to help define each subfamily.
we included the sole s. cerevisiae importin α, srp <dig>  in the tree to illustrate the relationships of the human kpna proteins to a common ancestor.
the phylogenetic tree shows that kpna <dig> is a member of the α <dig> subfamily.
the alignment of kpna <dig> with kpna's 1- <dig>  revealed that the ibb of kpna <dig> has a region of low identity to the consensus.
separate phylogenetic analysis of the arm repeats and ibb of the kpnas show that the kpna <dig> ibb is more divergent than the body of kpna <dig> , as the branch length separating the kpna <dig> ibb from s. cerevisiae importin α is longer than the branch length to the kpna <dig> body.
the α <dig> subfamily has the least divergent body, while the ibbs of this subfamily are almost as divergent as the kpna <dig> ibb.
kpna <dig>  kpna <dig>  and kpna <dig> show the same branch lengths for both their bodies and ibb's, suggesting they evolved at the same rate.
kpna <dig> has an n-terminal domain which is 48% identical to the kpna <dig> ibb, and only 24% identical to the kpna <dig> ibb.
the strong difference in the ibb of kpna <dig> from all other kpnas suggests that the kpna <dig> ibb is has diverged in response to different selective pressures.
the n-terminus of kpna <dig> is a functional ibb
importin α proteins act as adaptors for nuclear import by binding both nls cargo and importin β. importin α binding to importin β is mediated by the n terminal ibb domain, making possible the α/β heterodimer capable of carrying nls cargo into the nucleus.
the low sequence identity of the kpna <dig> ibb with other kpna ibb domains could be indicative of changes in function, and so we tested whether the n-terminus of kpna <dig> acts as an ibb.
we examined kpna <dig> binding to importin β by immobilizing gst-importin β on glutathione beads, and incubating the beads with in vitro translated, 35s-methionine labeled kpna proteins .
surprisingly, kpna <dig> displayed the most stable binding to the gst-importin β. a longer exposure of the film reveals a low level of kpna1- <dig> binding to importin β. because the autoinhibited state and importin β binding are mutually exclusive, the low level of kpna1- <dig> binding to importin β likely reflects the stability of the autoinhibited state of these proteins.
in order to confirm that the β binding of kpna <dig> is conferred by its ibb region, we performed a gst pulldown assay using in vitro translated, 35s-methionine labeled proteins; kpna <dig>  and a mutant kpna <dig> in which the putative ibb had been removed .
deletion of the kpna <dig> ibb resulted in complete loss of kpna <dig> binding to importin β .
because reticulocyte lystate contains proteins  that might influence the outcome of these binding assays, we used recombinant proteins to formally test whether the n-terminus of kpna <dig> functions as an ibb.
gst fusions of the ibb domains from kpna <dig> and kpna <dig> were immobilized on glutathione beads, recombinant importin β was added, and bound fractions analyzed by immunoblotting.
importin β bound to the ibb domains of both kpna <dig> and kpna7; the interactions were ran-sensitive because they were reduced significantly by including ranq69l  in the binding reaction .
we also found that preformed complexes of importin β with ibb domains of kpna <dig> and kpna <dig> were dissociated by addition of rangtp , a reaction that mimics ran-dependent disassembly of import complexes.
based on semi-quantitative immunoblotting, rangtp addition resulted in dissociation of 60% of the preformed importin β-kpna <dig> complexes but only 3% of the preformed importin β- kpna <dig> complexes.
this result, together with data showing that full-length kpna <dig> binds efficiently to importin β , suggests that kpna <dig> import complexes could be more stable than complexes formed between importin β and other kpna proteins.
the competitive autoinhibition of nls binding by the ibb may be altered by changes in the ibb affinity for importin α. thus we decided to test whether the kpna7Δibb would show enhanced binding to the sv40-nls compared to full length kpna <dig>  unexpectedly, neither full length nor ibb deletion forms of kpna <dig> display significant levels of binding to the sv <dig> nls .
the difference in sv <dig> nls binding affinities between kpna <dig> or kpna  <dig> and kpna <dig> suggests that there may be a difference in their nls binding surfaces.
crystal structures have been solved for several importin α proteins, yeast srp <dig>  <cit> , mouse and human kpna <dig>  <cit> , and human kpna <dig>  <cit> .
the network of amino acids that create the nls binding surface have been described in detail for the sv <dig> nls, rb nls, and nucleoplasmin  nls, among others  <cit> .
we used molecular modeling to examine the conservation of the nls binding surface between kpna <dig> and kpna <dig>  we mapped identical and conserved residues between kpna <dig> and kpna <dig>  as well as kpnas  <dig> through  <dig>  onto the structure of mouse kpna <dig> co-crystalized with the bipartite rb nls .
the rb nls binds both the major and minor binding sites on importin α, and thus serves as a reference for the entire nls binding surface  <cit> .
the residues in kpna <dig> that bind the rb nls are identical in kpna <dig>  moreover, the nls binding surface is almost perfectly conserved in all seven kpna proteins.
22/ <dig> amino acids that interact with the rb nls  <cit>  and 16/ <dig> which interact with the sv <dig> nls  <cit>  are perfectly conserved among the human kpna proteins .
the only amino acid involved in sv <dig> nls binding that is not identical in all family members is r <dig> in kpna <dig>  which corresponds to k <dig> ; s <dig> ; s <dig> ; k <dig> ; k <dig> ; and q <dig> .
notably, conservation at this position is observed within the α <dig> and α <dig> subfamilies.
kpna  <dig> shows limited rb nls binding and fails to bind to either the sv <dig> nls or the nucleoplasmin  nls
kpna <dig>  kpna <dig>  kpna <dig>  kpna <dig>  and kpna <dig> import cargo containing the sv <dig> nls in permeabilized cell assays  <cit> .
the perfect conservation of amino acids that form the rb nls binding surface on kpnas  <dig> through  <dig> suggests these receptors should have similar affinities for rb nls.
to examine the rb nls and sv <dig> nls binding characteristics of the entire importin α family, we performed binding assays with in vitro translated 35s methionine-labeled kpna proteins .
the target nls proteins were expressed as gst fusions and bound to glutathione beads.
kpna <dig> and kpna <dig>  bound to the rb nls weakly, while kpna <dig>  kpna <dig>  and kpna <dig>  bound the rb nls at a high level.
the sv <dig> binding was more complex, in that it did not track with subfamily, as kpna <dig>  kpna <dig>  kpna <dig>  and kpna <dig> bound the sv <dig> nls strongly; kpna <dig> bound less well; and kpna <dig> and kpna <dig> failed to bind the sv <dig> nls altogether.
notably, rb nls binding appears to be consistent within subfamilies, while the sv <dig> nls binding showed binding differences within subfamilies.
after we deposited the human kpna <dig> sequence to genbank , tejomurtula et al.
reported the sequence of bovine kpna <dig> and showed that it binds bovine nucleoplasmin  <cit> .
we therefore tested the ability of human kpna <dig> to bind the human npm nls  .
the npm nls bound to kpna <dig> very strongly, and showed some binding to kpna <dig>  kpna <dig>  and kpna <dig> .
under our reaction conditions, neither kpna <dig> nor knpa7Δibb bound to the human npm nls.
kpna <dig> localizes to the nucleus in hela cells
the steady state cellular localization of kpna <dig> is predominantly cytoplasmic, a distribution that represents the steady state balance between importin α import into the nucleus by importin β, and recycling to the cytoplasm by cas.
in order to examine the steady state localization of knpa <dig>  we generated an affinity-purified antibody to a unique sequence in its c-terminus .
double-label immunofluorescence microscopy was performed on hela cells with the kpna <dig> polyclonal antibody and a kpna <dig> monoclonal antibody.
this revealed that kpna <dig> is predominantly nuclear under conditions where kpna <dig> is largely cytoplasmic .
by immunoblotting, the kpna <dig> antibody detects a  <dig> kda protein, which is close to the predicted molecular weight of kpna <dig> .
preincubating the kpna <dig> antibody eliminates reactivity on immunoblots  and by immunofluorescence microscopy .
to independently assess the localization of these receptors, we transfected hela cells with plasmids containing hemagglutinin tagged forms of kpna <dig> and kpna <dig>  consistent with the results obtained with the endogenous proteins, ha-tagged kpna <dig> and kpna <dig> localized to the cytosol and nucleus, respectively .
discussion
here we describe the characterization of a new member of the human importin α family, kpna <dig>  sequence analysis revealed that kpna <dig> is most similar to kpna <dig> , and that it is the most divergent member of the importin α family.
sequence divergence in kpna <dig> occurs throughout the protein, including the ibb domain.
our binding data indictates that the ibb of kpna <dig> binds importin β stronger than other kpna proteins.
the nls binding surface is highly conserved in all of the importin α family members, but we observed clear differences among the different family members for binding the rb nls, sv <dig> nls, and the npm nls.
kpna <dig> bound to the rb nls with equivalent affinity to knpa <dig>  but failed to bind both the sv <dig> nls and the npm nls.
in addition, by immunofluorescence microscopy kpna <dig> localizes to the nucleus, a distribution that contrasts with the predominantly cytoplasmic localization of kpna <dig>  our data emphasizes that despite a high degree of structural conservation, there are important differences in the nls binding specificity and nucleocytoplasmic trafficking of importin -a family members.
based on the conservation of the residues which make up the rb nls and sv <dig> nls binding surfaces of kpna <dig>  we reached the conclusion that the nls binding surface is almost perfectly conserved in all seven members of the importin α family.
it was surprising, therefore to observe kpna-specific binding to both the rb nls and sv <dig> nls.
differences in the ibbs of importin α family members could have potentially altered the affinity for nls binding through autoinhibition  <cit> , but deletion of the ibb from kpna <dig> did not alter its affinity for either the sv <dig> nls or the npm nls.
assignment of the conserved residues is based upon the clustalw alignment of the alpha family members, which was then superimposed upon the crystal structure of mouse kpna <dig>  the key residues are perfectly conserved, but this method of examining the conservation does not take into account gaps, deletions, or the effects of substitutions outside the binding surface on the ultimate structure of the binding surface.
there is variability between α family members in the rest of their structure that may result in slight changes in the shape of the binding surface or relative location of the conserved residues which may then alter the nls specificity of a given importin α. evidence that the protein context of an nls plays a role in the specificity for a particular importin α  <cit>  suggests that an otherwise generic nls may acquire specificity for a specific importin α due to structural constraints imposed by its protein context.
subtle differences in the conformation of the nls binding surface may therefore lead to specificity for a contextualized nls, and promiscuous binding to an unstructured nls encountered out of its proper protein context.
these results emphasize the need for experimental validation of predictions made for paralogues.
understanding how highly similar members of the same protein family achieve nls selective binding will require structural analysis.
kpna <dig> is the most divergent of the seven proteins in the human importin α family, and the ibb domain is more divergent than the arm repeats, which maintain identity in the amino acids necessary for interaction with both the rb nls and the sv <dig> nls.
the lack of conservation in the kpna <dig> ibb suggests that it does not have the same selective pressures as kpnas  <dig> through  <dig>  there are two known functions for the ibb, binding to importin β, and autoinhibition through binding to importin α. importin β binding could be affected by the amino acid changes in the kpna <dig> n-terminus, but our data show very strong binding to importin β. this increased binding to importin β may represent an enhanced affinity of the ibb for importin β, but it could also be due to decreased autoinhibitory binding of the ibb to importin α.
a striking feature of kpna <dig> is its localization to the nucleus under steady state conditions, while kpna <dig> is predominantly cytoplasmic.
this nuclear localization suggests that nucleocytoplasmic shuttling of kpna <dig> differs from that of the other members of the importin α family.
there are three possibilities for the altered subcellular distribution; 1) enhanced nuclear import by importin β; 2) decreased nuclear export by cas; and 3) inefficient release of kpna <dig> from target nls, resulting in nuclear retention.
the most likely of these situations, given our evidence that kpna <dig> binds importin β with much higher affinity than other a family members is that this enhanced binding results in increased nuclear import of kpna <dig>
the recent study on bovine kpna <dig> showed that it is expressed preferentially in the embryo, suggesting a possible role in early in development  <cit> .
given that kpna <dig> plays a critical role in neuronal differentiation  <cit> , restricted expression of kpna isoforms could provide a mechanism for selective import of factors that drive key events during development.
CONCLUSIONS
we identified and characterized kpna <dig>  a highly divergent member of the importin a family of nuclear import receptors.
the importin α family in vertebrates comprises seven proteins, kpna <dig> through kpna <dig>  sequence analysis and molecular modeling revealed the residues that form the nls binding surface are nearly perfectly conserved throughout the importin a family.
nonetheless, there are striking differences in nls recognition amongst the different kpna proteins.
tertiary structure in kpna proteins could play a major role in creating binding surfaces that can discriminate between different nls's.

 eukaryotic cells are defined by the separation of dna from the rest of the cell by the nuclear envelope, a double bilayer made selectively permeable by nuclear pore complexes. transport of proteins between the nucleus and the cytoplasm is carried out by karyopherins, a family of proteins made up of importins and exportins. classical nuclear localization signal dependent nuclear import is carried out by importin α and importin β importin α family members bind nls cargo, and bind to importin β through an n-terminal importin β binding domain. importin β mediates translocation of the nls-importin α-importin β import complex into the nucleus through direct interactions with the npc. once in the nucleus, rangtp binds to importin β and induces dissociation of the import complex. exportin mediated nuclear export is regulated by rangtp through a related mechanism. whereas rangtp dissociates import complexes by binding importins, exportins must bind to rangtp in order to bind nuclear export signal containing cargoes. the heterotrimeric export complex then translocates through the npc and is dissociated in the cytoplasm by rangap stimulated conversion of rangtp to rangdp. while there are at least importin β family members which can bind directly to cargo and mediate import, importin β is unique in its ability to bind the importin α family of nuclear transport receptors. importin α binds to two major classes of nls, both characterized by basic amino acids; a monopartite nls, such as the sv nls, which consists of a single cluster of basic amino acids; and a bipartite nls, such as the retinoblastoma nls, which consists of two clusters of basic amino acids, separated by a ~ residue spacer. the architecture of importin α proteins is composed of armadillo repeats, a three a-helix motif named for the d. melanogaster homologue of β catenin. the binding site for a monopartite nls is located between the 2nd and 4th arm repeats and is called the major site. importin α binds to the c-terminus of bipartite nls sequences with the major site and to the n-terminal element of the bipartite nls using a smaller site created by the 7th and 8th arm repeats called the minor site. the accessibility of these nls binding sites is regulated by an autoinhibitory mechanism. the ibb of importin α contains basic amino acids that bind to the nls binding surface when the receptor is in an autoinhibited state. importin α binding to nls cargo and to importin β is, therefore, a cooperative process because importin β binding to the ibb relieves the autoinhibition of importin α. relief of autoinhibition facilitates importin α binding to nls cargo. after nuclear import, the complex is dissociated by the cooperative effects of rangtp binding to importin β, and binding of importin α to cas. cas is an exportin which forms a trimeric complex consisting of cas, rangtp and importin α, and is responsible for recycling importin α to the cytoplasm. yeasts encode a single importin α, but higher eukaryotes encode three importin α subfamilies, designated importin α α and α there are six previously described human importin α forms, each encoded by different gene. importin α family members show preferences for specific types of nls cargo, although there is also some functional redundancy. most nlss are very similar in sequence leading to the suggestion that importin α specificity is conferred by the protein context of the nls imparted by the cargo. it appears that additional importin α proteins are required for a multicellular organism because different importin α's have specific cargoes to import during differentiation. studies in d. melanogaster have shown that importin α is not required in adult flies, but is required for oogenesis and that importin α is favored for a step in larval development. similarly, it has been shown in c. elegans that while importin α is ubiquitously expressed, α and α are expressed only in the germline, and are not sufficient to replace α in somatic cells. in mouse embryonic stem cells, switching from importin α expression to importin α expression has been shown to induce neural differentiation. these data suggest that cargo interactions with specific importin α isotypes are important for controlling differentiation pathways. here we describe a novel importin α in humans, the seventh member of the importin α family, which we term karyopherin alpha. this new α family member is most similar to kpna and a member of the α subfamily. we show that kpna binds to importin β through an n-terminal ibb, but that its importin β binding affinity appears to be much higher than that of the other kpnas. kpna localization is predominantly nuclear in hela cells, highlighting another difference between and the other importin α family members.  kpna is a novel member of the importin alpha family there are six known members of the human importin α family. we used blast to query the human genome with the importin α family member kpna and retrieved a sequence predicted to encode a seventh member of the importin α family. because the sequence of the putative importin α was generated by gene prediction, we set out to experimentally validate the expression and function of the predicted importin α gene as an importin α. using rt-pcr, we amplified a product of the expected size from rna prepared from the prostate cancer cell line lncap. we sequenced the cdna, and found that it matched that of the predicted gene, leading us to conclude that the gene is transcribed in human cells. this gene is the seventh member of the importin α family of transport receptors, therefore we named it karyopherin alpha. the complete human importin α family is shown in table with their previous designations, as well as accession and gene id numbers. alignment of the kpna protein sequence with kpna's 1- using clustalw reveals that kpna is % identical to its closest importin α family member, the prototypical kpna. the maximum identity between kpna and members of the α and α subfamilies is only %. kpna kpna and kpna share a minimum of % identity, while kpna and kpna are % identical, emphasizing that kpna is highly divergent from other importin α family members. the multiple alignment shows, however, that kpna is related to other importin α family members throughout the length of the protein, suggesting that it is an nls binding protein. the number shown is the percent identity between the respective kpna proteins. here we show the kpna numbering system and the corresponding importin alpha numbers, as well as unique identifiers: accession number, gene id number, and the chromosomal locus. kpna homologues are found in vertebrates including mouse, xenopus and zebra fish. in all animals, importin α proteins are classified into subfamilies, α α and α. while vertebrates have seven importin α family members, invertebrates such as drosophila have only importin α proteins, one for each subfamily. we created a phylogenetic tree of the complete human importin α family using protdist. the drosophila melanogaster importin α proteins were included in the phylogram to help define each subfamily. we included the sole s. cerevisiae importin α, srp in the tree to illustrate the relationships of the human kpna proteins to a common ancestor. the phylogenetic tree shows that kpna is a member of the α subfamily. the alignment of kpna with kpna's 1- revealed that the ibb of kpna has a region of low identity to the consensus. separate phylogenetic analysis of the arm repeats and ibb of the kpnas show that the kpna ibb is more divergent than the body of kpna, as the branch length separating the kpna ibb from s. cerevisiae importin α is longer than the branch length to the kpna body. the α subfamily has the least divergent body, while the ibbs of this subfamily are almost as divergent as the kpna ibb. kpna kpna and kpna show the same branch lengths for both their bodies and ibb's, suggesting they evolved at the same rate. kpna has an n-terminal domain which is 48% identical to the kpna ibb, and only 24% identical to the kpna ibb. the strong difference in the ibb of kpna from all other kpnas suggests that the kpna ibb is has diverged in response to different selective pressures. the n-terminus of kpna is a functional ibb importin α proteins act as adaptors for nuclear import by binding both nls cargo and importin β. importin α binding to importin β is mediated by the n terminal ibb domain, making possible the α/β heterodimer capable of carrying nls cargo into the nucleus. the low sequence identity of the kpna ibb with other kpna ibb domains could be indicative of changes in function, and so we tested whether the n-terminus of kpna acts as an ibb. we examined kpna binding to importin β by immobilizing gst-importin β on glutathione beads, and incubating the beads with in vitro translated, 35s-methionine labeled kpna proteins. surprisingly, kpna displayed the most stable binding to the gst-importin β. a longer exposure of the film reveals a low level of kpna1- binding to importin β. because the autoinhibited state and importin β binding are mutually exclusive, the low level of kpna1- binding to importin β likely reflects the stability of the autoinhibited state of these proteins. in order to confirm that the β binding of kpna is conferred by its ibb region, we performed a gst pulldown assay using in vitro translated, 35s-methionine labeled proteins; kpna and a mutant kpna in which the putative ibb had been removed. deletion of the kpna ibb resulted in complete loss of kpna binding to importin β. because reticulocyte lystate contains proteins that might influence the outcome of these binding assays, we used recombinant proteins to formally test whether the n-terminus of kpna functions as an ibb. gst fusions of the ibb domains from kpna and kpna were immobilized on glutathione beads, recombinant importin β was added, and bound fractions analyzed by immunoblotting. importin β bound to the ibb domains of both kpna and kpna7; the interactions were ran-sensitive because they were reduced significantly by including ranq69l in the binding reaction. we also found that preformed complexes of importin β with ibb domains of kpna and kpna were dissociated by addition of rangtp, a reaction that mimics ran-dependent disassembly of import complexes. based on semi-quantitative immunoblotting, rangtp addition resulted in dissociation of 60% of the preformed importin β-kpna complexes but only 3% of the preformed importin β- kpna complexes. this result, together with data showing that full-length kpna binds efficiently to importin β, suggests that kpna import complexes could be more stable than complexes formed between importin β and other kpna proteins. the competitive autoinhibition of nls binding by the ibb may be altered by changes in the ibb affinity for importin α. thus we decided to test whether the kpna7Δibb would show enhanced binding to the sv40-nls compared to full length kpna unexpectedly, neither full length nor ibb deletion forms of kpna display significant levels of binding to the sv nls. the difference in sv nls binding affinities between kpna or kpna and kpna suggests that there may be a difference in their nls binding surfaces. crystal structures have been solved for several importin α proteins, yeast srp, mouse and human kpna, and human kpna. the network of amino acids that create the nls binding surface have been described in detail for the sv nls, rb nls, and nucleoplasmin nls, among others. we used molecular modeling to examine the conservation of the nls binding surface between kpna and kpna we mapped identical and conserved residues between kpna and kpna as well as kpnas through onto the structure of mouse kpna co-crystalized with the bipartite rb nls. the rb nls binds both the major and minor binding sites on importin α, and thus serves as a reference for the entire nls binding surface. the residues in kpna that bind the rb nls are identical in kpna moreover, the nls binding surface is almost perfectly conserved in all seven kpna proteins. 22/ amino acids that interact with the rb nls and 16/ which interact with the sv nls are perfectly conserved among the human kpna proteins. the only amino acid involved in sv nls binding that is not identical in all family members is r in kpna which corresponds to k; s; s; k; k; and q. notably, conservation at this position is observed within the α and α subfamilies. kpna shows limited rb nls binding and fails to bind to either the sv nls or the nucleoplasmin nls kpna kpna kpna kpna and kpna import cargo containing the sv nls in permeabilized cell assays. the perfect conservation of amino acids that form the rb nls binding surface on kpnas through suggests these receptors should have similar affinities for rb nls. to examine the rb nls and sv nls binding characteristics of the entire importin α family, we performed binding assays with in vitro translated 35s methionine-labeled kpna proteins. the target nls proteins were expressed as gst fusions and bound to glutathione beads. kpna and kpna bound to the rb nls weakly, while kpna kpna and kpna bound the rb nls at a high level. the sv binding was more complex, in that it did not track with subfamily, as kpna kpna kpna and kpna bound the sv nls strongly; kpna bound less well; and kpna and kpna failed to bind the sv nls altogether. notably, rb nls binding appears to be consistent within subfamilies, while the sv nls binding showed binding differences within subfamilies. after we deposited the human kpna sequence to genbank, tejomurtula et al. reported the sequence of bovine kpna and showed that it binds bovine nucleoplasmin. we therefore tested the ability of human kpna to bind the human npm nls. the npm nls bound to kpna very strongly, and showed some binding to kpna kpna and kpna. under our reaction conditions, neither kpna nor knpa7Δibb bound to the human npm nls. kpna localizes to the nucleus in hela cells the steady state cellular localization of kpna is predominantly cytoplasmic, a distribution that represents the steady state balance between importin α import into the nucleus by importin β, and recycling to the cytoplasm by cas. in order to examine the steady state localization of knpa we generated an affinity-purified antibody to a unique sequence in its c-terminus. double-label immunofluorescence microscopy was performed on hela cells with the kpna polyclonal antibody and a kpna monoclonal antibody. this revealed that kpna is predominantly nuclear under conditions where kpna is largely cytoplasmic. by immunoblotting, the kpna antibody detects a kda protein, which is close to the predicted molecular weight of kpna. preincubating the kpna antibody eliminates reactivity on immunoblots and by immunofluorescence microscopy. to independently assess the localization of these receptors, we transfected hela cells with plasmids containing hemagglutinin tagged forms of kpna and kpna consistent with the results obtained with the endogenous proteins, ha-tagged kpna and kpna localized to the cytosol and nucleus, respectively. discussion here we describe the characterization of a new member of the human importin α family, kpna sequence analysis revealed that kpna is most similar to kpna, and that it is the most divergent member of the importin α family. sequence divergence in kpna occurs throughout the protein, including the ibb domain. our binding data indictates that the ibb of kpna binds importin β stronger than other kpna proteins. the nls binding surface is highly conserved in all of the importin α family members, but we observed clear differences among the different family members for binding the rb nls, sv nls, and the npm nls. kpna bound to the rb nls with equivalent affinity to knpa but failed to bind both the sv nls and the npm nls. in addition, by immunofluorescence microscopy kpna localizes to the nucleus, a distribution that contrasts with the predominantly cytoplasmic localization of kpna our data emphasizes that despite a high degree of structural conservation, there are important differences in the nls binding specificity and nucleocytoplasmic trafficking of importin -a family members. based on the conservation of the residues which make up the rb nls and sv nls binding surfaces of kpna we reached the conclusion that the nls binding surface is almost perfectly conserved in all seven members of the importin α family. it was surprising, therefore to observe kpna-specific binding to both the rb nls and sv nls. differences in the ibbs of importin α family members could have potentially altered the affinity for nls binding through autoinhibition, but deletion of the ibb from kpna did not alter its affinity for either the sv nls or the npm nls. assignment of the conserved residues is based upon the clustalw alignment of the alpha family members, which was then superimposed upon the crystal structure of mouse kpna the key residues are perfectly conserved, but this method of examining the conservation does not take into account gaps, deletions, or the effects of substitutions outside the binding surface on the ultimate structure of the binding surface. there is variability between α family members in the rest of their structure that may result in slight changes in the shape of the binding surface or relative location of the conserved residues which may then alter the nls specificity of a given importin α. evidence that the protein context of an nls plays a role in the specificity for a particular importin α suggests that an otherwise generic nls may acquire specificity for a specific importin α due to structural constraints imposed by its protein context. subtle differences in the conformation of the nls binding surface may therefore lead to specificity for a contextualized nls, and promiscuous binding to an unstructured nls encountered out of its proper protein context. these results emphasize the need for experimental validation of predictions made for paralogues. understanding how highly similar members of the same protein family achieve nls selective binding will require structural analysis. kpna is the most divergent of the seven proteins in the human importin α family, and the ibb domain is more divergent than the arm repeats, which maintain identity in the amino acids necessary for interaction with both the rb nls and the sv nls. the lack of conservation in the kpna ibb suggests that it does not have the same selective pressures as kpnas through there are two known functions for the ibb, binding to importin β, and autoinhibition through binding to importin α. importin β binding could be affected by the amino acid changes in the kpna n-terminus, but our data show very strong binding to importin β. this increased binding to importin β may represent an enhanced affinity of the ibb for importin β, but it could also be due to decreased autoinhibitory binding of the ibb to importin α. a striking feature of kpna is its localization to the nucleus under steady state conditions, while kpna is predominantly cytoplasmic. this nuclear localization suggests that nucleocytoplasmic shuttling of kpna differs from that of the other members of the importin α family. there are three possibilities for the altered subcellular distribution; 1) enhanced nuclear import by importin β; 2) decreased nuclear export by cas; and 3) inefficient release of kpna from target nls, resulting in nuclear retention. the most likely of these situations, given our evidence that kpna binds importin β with much higher affinity than other a family members is that this enhanced binding results in increased nuclear import of kpna the recent study on bovine kpna showed that it is expressed preferentially in the embryo, suggesting a possible role in early in development. given that kpna plays a critical role in neuronal differentiation, restricted expression of kpna isoforms could provide a mechanism for selective import of factors that drive key events during development.  we identified and characterized kpna a highly divergent member of the importin a family of nuclear import receptors. the importin α family in vertebrates comprises seven proteins, kpna through kpna sequence analysis and molecular modeling revealed the residues that form the nls binding surface are nearly perfectly conserved throughout the importin a family. nonetheless, there are striking differences in nls recognition amongst the different kpna proteins. tertiary structure in kpna proteins could play a major role in creating binding surfaces that can discriminate between different nls's. 
kpna shows a predominantly nuclear distribution under steady state conditions, which contrasts with kpna which is primarily cytoplasmic. classical nuclear localization signal dependent nuclear import is carried out by a heterodimer of importin α and importin β. nls cargo is recognized by importin α, which is bound by importin β. importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, rangtp binding to importin β triggers disassembly of the complex. to date, six importin α family members, encoded by separate genes, have been described in humans. we sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α, which is most closely related to kpna the domain of kpna that binds importin β is divergent, and shows stronger binding to importin β than the ibb domains from of other importin α family members. kpna is a novel importin α family member in humans that belongs to the importin α subfamily. kpna shows different subcellular localization and nls binding characteristics compared to other members of the importin α family.
 classical nuclear localization signal dependent nuclear import is carried out by a heterodimer of importin α and importin β. nls cargo is recognized by importin α, which is bound by importin β. importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, rangtp binding to importin β triggers disassembly of the complex. to date, six importin α family members, encoded by separate genes, have been described in humans.  we sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α, which is most closely related to kpna the domain of kpna that binds importin β is divergent, and shows stronger binding to importin β than the ibb domains from of other importin α family members. with regard to nls recognition, kpna binds to the retinoblastoma nls to a similar degree as kpna but it fails to bind the sv40-nls and the human nucleoplasmin nls. kpna shows a predominantly nuclear distribution under steady state conditions, which contrasts with kpna which is primarily cytoplasmic.  kpna is a novel importin α family member in humans that belongs to the importin α subfamily. kpna shows different subcellular localization and nls binding characteristics compared to other members of the importin α family. these properties suggest that kpna could be specialized for interactions with select nls-containing proteins, potentially impacting developmental regulation. 