Karyopherinβ proteins (Kapßs; also known as Importins and Exportins) are responsible for the majority of nucleo-cytoplasmic transport in the cell. At least 20 members of the Kapβ family have been identified in humans. Kapßs bind specific sets of transport substrates and target them to the nuclear pore complex. The Ran GTPase regulates Kapβ-substrate interactions and transport directionality through its nucleotide cycle (Chook and Blobel, 2001
; Conti and Izaurralde, 2001
; Gorlich and Kutay, 1999
; Weis, 2003
). RanGTP is concentrated in the nucleus, while RanGDP is concentrated in the cytoplasm. In import pathways, RanGTP and substrates bind Kapßs competitively, allowing substrate binding in the cytoplasm and RanGTP-mediated release in the nucleus. In contrast, in export pathways, RanGTP, substrates, and Kapßs bind cooperatively, resulting in substrate binding in the nucleus and release in the cytoplasm as the Ran bound nucleotide is hydrolyzed.
In humans, ten import Kapßs have been shown to carry a diverse set of macromolecular substrates into the nucleus (Mosammaparast and Pemberton, 2004
). Despite significant efforts, only a few substrates have been identified for most import Kapßs, and large panels of substrates have been identified for only two pathways: those of Kapβ1 and Kapβ2 (see below). Each import Kapβ appears to bind distinct sets of substrates, suggesting that each Kapβ recognizes a different nuclear localization signal(s) (NLS[s]). However, large sequence diversity among various substrates has prevented identification of NLSs for most Kapßs, and it remains extremely difficult to predict NLSs in candidate import substrates.
The classical NLSs are short, lysine-rich sequences that bind the adaptor protein Kapα, which forms a heterodimer with Kapβ1, which in turn mediates nuclear import (Conti and Izaurralde, 2001
). Most other proteins imported into the nucleus do not utilize such an adaptor but rather bind directly to a Kapβ. The few characterized NLSs that bind directly to Kapßs are diverse, encompassing both structural domains and linear epitopes. For example, crystal structures of three Kapβ1-substrate complexes show structurally diverse substrates binding at different sites on the karyopherin (Cingolani et al., 1999
; Lee et al., 2003
). Furthermore, most proteins that bind Kapβ1 show little sequence or structural homology, and thus general features among substrates in this pathway cannot be inferred at this time.
In another import pathway, more than 20 mRNA processing proteins (including hnRNPs A1, D, F, M, HuR, DDX3, Y-box binding protein 1, and TAP) have been identified as import substrates of Kapβ2 (Bonifaci et al., 1997
; Fan and Steitz, 1998
; Guttinger et al., 2004
; Kawamura et al., 2002
; Pollard et al., 1996
; Rebane et al., 2004
;Siomi et al., 1997
; Suzuki et al., 2005
; Truant et al., 1999
). Kapβ2 binds its best-characterized substrate, splicing factor hnRNP A1, through the 38 residue M9 sequence (Bonifaci et al., 1997
; Pollard et al., 1996
) that we will refer to as M9NLS. Many studies have shown that the M9NLS peptide is both necessary and sufficient for nuclear import mediated by Kapβ2 (Siomi and Dreyfuss, 1995
; Weighardt et al., 1995
). Other than hnRNP A1, only NLSs in HuR (Fan and Steitz, 1998
), TAP (Truant et al., 1999
), and hnRNP D and its homologs, the JKTPB proteins (Kawamura et al., 2002
; Suzuki et al., 2005
), have been characterized. The NLSs of hnRNP D and HuR show marginal sequence homology to M9NLS, that of TAP shares no sequence homology with M9NLS, and none of the other Kapβ2 substrates contain obviousM9NLS-like sequences. Like the Kapβ1 system, the diversity of substrates and known NLSs in Kapβ2 has also prevented prediction of NLSs in this pathway.
In the nucleus, RanGTP binds import Kapßs with high affinity and dissociates substrates (Chook et al., 2002
;Floer and Blobel, 1996
; Gorlich et al., 1996
). The unique repertoire of substrates for individual Kapßs suggests significant differences in their mechanisms of substrate recognition and therefore also differences in their regulation by Ran. The latter is illustrated in two different models for Ran-mediated substrate dissociation in the Kapβ1 and Kapβ2 pathways. For structurally diverse Kapβ1 substrates that also bind at different sites on the karyopherin, Ran-mediated dissociation involves both a global conformational change that locks the Kapβ1 superhelix into a substrate-incompatible conformation and a direct displacement by Ran (Cingolani et al., 1999
; Lee et al., 2003
; Vetter et al., 1999
). Alternatively, structural and biochemical analyses of the Kapβ2-RanGTP complex suggest that RanGTP and substrate binding sites do not overlap and that an internal loop of Kapβ2 is crucial for substrate dissociation in the presence of Ran (Chook and Blobel, 1999
; Chook et al., 2002
). Thus it appears that the two best-known nuclear import pathways may utilize Ran to dissociate substrates in different manners.
In order to understand the mechanism of substrate recognition and distill the critical elements for NLS recognition by Kapβ2, and to understand the mechanism of Ran-mediated substrate dissociation for this import pathway, we have determined the structure of Kapβ2 bound to the M9NLS of hnRNP A1. The structure and complementary biochemical studies reveal a set of rules for NLS recognition by Kapβ2: NLSs imported by Kapβ2 should occur within large (>30-residue) structurally disordered elements, have overall basic character, and contain a set of consensus sequences. These rules are predictive and have allowed us to identify and biochemically confirm NLSs in seven known Kapβ2 substrates. Most importantly, we used these NLS rules in a bioinformatics approach and identified 81 new candidate import substrates for Kapβ2. We have confirmed that five of these bind Kapβ2 through the predicted NLS in a Ran-dependent manner. Finally, comparison with the previously determined structure of the Kapβ2-Ran complex (Chook and Blobel, 1999
) has revealed the mechanism of Ran-mediated substrate dissociation. M9NLS binds in the C-terminal arch of Kapβ2, in a site spatially distinct from the Ran binding site. However, in the Ran complex, the acidic loop of Kapβ2 occupies this substrate binding site. Thus, Ran binding induces structural changes in Kapβ2 that are incompatible with substrate binding.