No abstract

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Importin-␣ is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-␣, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-␣ and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10 -12 KRRK) that can be used to identify novel nuclear proteins.Nucleocytoplasmic transport occurs through nuclear pore complexes, large proteinaceous structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The best characterized nuclear targeting signals are the conventional nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). The NLSs fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters separated by a spacer. Despite the variability, the conventional basic NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews, see Refs. 2-4). Importin-␣ (Imp␣) contains the NLSbinding site, and importin-␤ (Imp␤) is responsible for the translocation of the importin-substrate complex through the nuclear pore complex. Once inside the nucleus, Ran-GTP binds to Imp␤ and causes the dissociation of the import complex. Imp␣ becomes autoinhibited, and both importin subunits return to the cytoplasm separately without the import cargo. The directionality of nuclear import is conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Imp␣ consists of two structural and functional domains, a short basic N-terminal Imp␤-binding domain (5-7) and a large NLS-binding domain built of armadillo (Arm) repeats (8). The structural basis of monopartite and bipartite NLS recognition by Imp␣ has been studied crystallographically in yeast and mouse Imp␣ proteins (9 -11). The two basic clusters of the bipartite NLSs bind to two separate binding sites on Imp␣, involving Arm repeats 1-4 and 4 -8, respectivel...

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-␣/␤ heterodimer. Importin-␣ contains the NLS binding site, whereas importin-␤ mediates the translocation through the nuclear pore. We characterized the interactions involving importin-␣ during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-␣ is shown to exist in a monomeric autoinhibited state (as- Nucleocytoplasmic transport occurs through nuclear pore complexes, large supramolecular structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The first and best characterized nuclear targeting signals are the classical nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). NLSs do not conform to a specific consensus sequence and fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters.Despite the sequence variability, the classical NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews see Refs. 2-6). Importin-␣ contains the NLS-binding site, and importin-␤ is responsible for the translocation of the importincargo complex through the pore. Transfer through the pore is facilitated by the proteins Ran (Ras-related nuclear protein) and nuclear transport factor-2. Once inside the nucleus, importin-␤ binds to Ran-GTP to effect the dissociation of the import complex; the importin subunits return to the cytoplasm separately and without the cargo. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Importin-␣ consists of two structural and functional domains, a short basic N-terminal importin-␤-binding (IBB) domain (7-9), and a large NLS-binding domain comprising armadillo (Arm) repeats (10). The monopartite NLSs bind at a major site located between the first and fourth Arm repeats and additionally at a minor site spanning repeats 4 -8 (11-13). The bipartite NLSs span the two binding sites, with each site recognizing one of the basic clusters (12, 13). The linker sequence between the two basic clusters makes few contacts with the receptor, consistent with its tolerance to mutations. The affinity of the importin-targeting sequence interaction is a critical parameter in determining transport efficiency (3).The structure of mouse importin-␣ showed that in the absence of importin-␤ or NLS-containing proteins, a part of the IBB domain occupies the major NLS-binding site (14). Based on this observation, it was sugges...