Review
Karyopherins and nuclear import

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Abstract

Proteins of the karyopherin α and karyopherin β families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin α and its complexes with nuclear localization signal peptides, a karyopherin β2–Ran complex and complexes of full-length and fragments of karyopherin β1 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.

Introduction

In eukaryotic cells, signal-mediated macromolecular transport between the nucleus and the cytoplasm is an integral part of many processes, such as gene expression, signal transduction and cell-cycle progression. Nucleocytoplasmic transport occurs through cylindrical structures spanning the nuclear envelope known as nuclear pore complexes (NPCs). NPCs are large protein assemblies of approximately 125 MDa in mammalian cells and approximately 60 MDa in yeast. These structures allow passive exchange of ions, small molecules and small proteins (<20 KDa), but restrict passage of macromolecules to only those bearing appropriate signals. The direction of transport through the NPC is determined by a signal. The nuclear localization signal (NLS) directs proteins into the nucleus and the nuclear export signal (NES) directs the transport of proteins into the cytoplasm. Karyopherinα (Kapα—also known as importinα) is an adaptor protein that recognizes the first discovered or classical NLS, which is characterized by one or two stretches of basic residues [1]. Kapα also interacts with Kapβ1 (also known as importinβ) and together these proteins form a transport pathway in which proteins containing a classical NLS are imported by the Kapα–Kapβ1 heterodimer (Fig. 1; [1]). Over the past several years, many other import pathways have been identified, all involving transport substrates with NLSs distinct from the ‘classical’ sequence [1]. Nonclassical NLSs have diverse amino acid sequences that bind directly and specifically to the different Kapβ1 homologs that constitute the Kapβ family. Unlike the classical NLS, which interacts with Kapβ1 indirectly through an adaptor protein, almost all other NLSs bind directly to their specific Kapβs. There are 14Kapβs in yeast and more than 20 in mammalian cells. Members of the Kapβ family share similar molecular weights (90–150 kDa) and isoelectric points (4.0–5.0), and contain multiple tandem helical repeats termed HEAT repeats. Thirteen of the yeast Kapβs and a handful of the mammalian Kapβs have identified functions in nuclear import (some import karyopherins are also known as importins), nuclear export (export karyopherins are also known as exportins) or bidirectional nuclear transport. Recently, Kapα and Kapβ1 have also been shown to have important regulatory functions in mitosis as they act as ‘chaperones’ to sequester crucial factors for spindle formation, although this aspect of karyopherin function will not be reviewed here 2., 3., 4.. The Kapβ family is summarized in Table 1.

Kapβs interact with both the transport substrate and NPC proteins (nucleoporins), thus targeting the substrate to the NPC. Substrate–Kapβ interactions are regulated by the GTPase Ran; the mode of interaction between the three proteins and their response to the Ran nucleotide state are key determinants of transport direction. In nuclear import, complexation of Kapβs with Ran•GTP results in substrate dissociation in the nucleus (Fig. 1) 1., 5•., 6., 7., whereas interactions between export Kapβs, Ran•GTP and export substrates seem to be cooperative 1., 8., 9.. Like most GTPases, the nucleotide state of Ran is regulated by the Ran GTPase activating protein (RanGAP), which catalyzes nucleotide hydrolysis, and by the Ran guanine nucleotide exchange factor (RanGEF), which catalyzes nucleotide exchange 10., 11.. Other Ran-binding proteins, such as RanBP1 and its homologs, and p10/NTF2, also modulate the stability of the different nucleotide states of Ran 12., 13., 14.. The specific localization of RanGAP to the cytoplasm and the cytoplasmic fibers of the NPC probably leads to Ran being predominantly in its GDP state on the cytoplasmic side of the NPC and the resulting lack of Ran•GTP allows formation of Kapβ–import substrate complexes 10., 15.. Conversely, the nuclear localization of RanGEF to chromatin and the NPC nuclear basket forms a pool of nuclear Ran•GTP, which contributes to the completion of nuclear import through GTPase-mediated Kapβ–substrate dissociation in the nucleus 1., 11., 16.. Recent evidence suggests that, although Ran•GTP plays a major role in substrate dissociation in many import pathways, there exist additional means for the process in the nucleus. Pemberton et al. 17., 18. showed preferential release of the import substrate TATA-binding protein (TBP) from Kap114p in the presence of its TATA-containing double-stranded DNA nuclear targets. These findings of nuclear-target-aided substrate dissociation suggest an important role for the delivery of import complexes to specific sites in the nucleus, presumably via intranuclear transport pathways [19]. Another recently discovered mechanism of import substrate dissociation involves pseudosubstrate sequences in nucleoplasmic nucleoporins such as Nup98 and Nup153 that compete for the Kapβ2 substrate-binding site and probably aid in disassembling the Kapβ2–substrate complex 16., 20..

Key issues in nuclear import include substrate recognition, targeting of the transport complex to the NPC, vectorial movement through the NPC and substrate dissociation in the nucleus. In the past two years, several high-resolution structures of Kapα and Kapβs have been determined, and we devote this paper to review the insights revealed by the structures of these karyopherin complexes. Structures of free and ligated Kapα have revealed the chemical and geometric requirements for specific NLS binding. In addition, they have explained the structural basis of Kapα autoregulation 21••., 22••., 23••., 24••.. The structure of Kapβ1 bound to the N-terminal domain of Kapα extends our understanding of the classical Kapα–Kapβ1 transport pathway by explaining how a Kapα–NLS complex can bind Kapβ1 [25••]. More generally, the structure of Kapβ1 bound to the N-terminal domain of Kapα also lays the foundation for understanding direct Kapβ–substrate recognition in nonclassical import pathways as the first structure of a Kapβ–‘import substrate’ complex. Although the mechanism of the vectorial movement of transport complexes across the NPC is heavily debated, it is generally agreed that interactions between Kapβs and many of the nucleoporins throughout the entire length of the NPC play a prominent role. The structure of an N-terminal fragment of Kapβ1 complexed with a fragment of yeast nucleoporin Nsp1p provides a glimpse of one of the many transit intermediates [26••]. Finally, structures of the Kapβ2–Ran•GppNHp (GppNHp is a nonhydrolyzable analog of GTP) complex and of a fragment of Kapβ1 bound to the GTPase have provided a rationale for the specificity of both Kapβ1 and Kapβ2 for the GTP-bound state of Ran, and indicated mechanisms for GTPase-mediated substrate dissociation 27••., 28••.. The availability of structures for both Kapβ1 and Kapβ2 (also known as transportin) also enables a comparison of two members of the Kapβ family, initiating efforts toward understanding the different substrate and nucleoporin specificities of the many different Kapβ-mediated nuclear transport pathways.

Section snippets

Karyopherins consist of repeated helical units

The basic Kapα structure is a cylindrical superhelix consisting of ten armadillo (ARM) repeats (Fig. 1, Fig. 2) 21••., 22••.. ARM motifs were first identified in the gene product of the Drosophila melanogaster segment polarity gene armadillo and its human ortholog β-catenin [29]. An individual ARM repeat contains approximately 40amino acids, which form three helices—H1, H2 and H3. Consecutive ARM repeats are related by a translation of approximately 9Å and a rotation of approximately 30°. The

Kapα: receptor for the classical nuclear localization signal

Kapα is an adaptor protein that binds proteins containing classical NLSs and Kapβ1. Kapα is divided into three structural units: a central NLS-binding domain with ten ARM repeats, a small hydrophilic C-terminal domain of unknown function and a positively charged, autoinhibitory N-terminal domain that can bind either the ARM domain or Kapβ1 (Fig. 1). Kapα recognizes a variety of classical NLSs, such as the very basic monopartite SV40 T antigen NLS (PKKKRKV), the more hydrophobic monopartite

Kapβ1–Kapα complex: archetype for Kapβ–substrate interaction

Kapβ1 is unique among the Kapβ family in its use of Kapα as an adaptor for binding substrates that contain the ‘classical’ NLS [1]. Other members of the Kapβ family bind their substrates directly. Kapβ1 can also import substrates such as HIV Rev, GAL4, T-cell tyrosine phosphatase, cyclin B1 and parathyroid hormone-related protein (PTHrP) without binding Kapα (Table 1) [39]. Therefore, the structure of full-length Kapβ1 bound directly to its substrate, the N-terminal domain of Kapα (Kapα11–54),

Kapβ–Ran complexes: implications for substrate dissociation

Crystal structures of full-length Kapβ2 bound to Ran•GppNHp (Fig. 3b) [27••] and of the N-terminal fragment of Kapβ1 (Kapβ11–462; full-length Kapβ1 has 876 residues) bound to the GTPase (Fig. 3c) [28••] provide pictures of one of the final steps of nuclear import; Ran•GTP forms a high-affinity complex with Kapβs and import substrates are concomitantly displaced and released into the nucleus. Kapβ–Ran•GTP complexes have also been shown to be important for recycling of import Kapβs to the

Interactions between Kapβ and nucleoporin

One of the most important functional requirements for Kapβs in mediating vectorial nuclear transport is their ability to bind the NPC. The yeast NPC is approximately 60 MDa and contains oligomers of approximately 30 different nucleoporins, whereas the vertebrate NPC is approximately 125 MDa with multiple copies of a yet undetermined number of nucleoporins [49•]. The NPC is a cylindrical structure that spans the double membrane of the nuclear envelope. It exhibits an eightfold rotational

Mechanisms of vectorial translocation through the nucleoporin complex

Models of the mechanisms for vectorial movement through the NPC must explain several observations: signal-mediated transport; passive passage of small molecules; exclusion of large molecules devoid of NLS/NES; the vectorial nature of transport; observed kinetics of transport; and the observed electron density in the central channel. Early models include opening and closing of the 260Å central channel of the NPC by ATP-driven mechanochemical means in response to signal sequences [53]. However,

Comparisons of the karyopherin superhelices: implications of conformational flexibility

The conformational flexibility of Kapα and Kapβs appears to be central to the function of these transport factors. The construction of these proteins from repeated units probably contributes to their flexibility. Comparisons between the different Kapα and Kapβ crystal structures indicate differences in the superhelical path resulting from changes in the relative arrangements of individual ARM/HEAT repeat units. Superposition of yeast Kapα complexed with either the SV40 T antigen NLS or the

Conclusions

Structures of Kapα and Kapβ complexes have contributed tremendously to our understanding of the molecular basis of nuclear import, especially in the area of substrate recognition in the classical Kapα–Kapβ1 pathway and in the more general area of GTPase binding and substrate dissociation. However, our understanding of the structural requirements and substrate specificity for the many nonclassical Kapβ transport pathways is still minimal. Future structural characterization of nonclassical

Acknowledgements

Because of the review's focus on structures of karyopherin complexes, we apologize for not including an extensive list of nonstructural references. We thank Thomas Schwartz for advice on figures and Mike Rosen for critical reading of the manuscript.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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