Crystallization of the flexible nuclear import receptor importin-β in the unliganded state (original) (raw)
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Dynamic localization of the nuclear import receptor and its interactions with transport factors
The Journal of Cell Biology, 1996
Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rnal-1 strain, we have identified a genetic interaction between Rnalp, a GTPase activating protein required for nuclear transport, and yeast importin-[3, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rnalp physically interacts with importin-~. Mutants in importin-~ exhibit in vivo nuclear protein import defects, and importin-[3 localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-ot, but not importin-13, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-a and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-a from the nucleus.
Importin-beta-like nuclear transport receptors
Genome biology, 2001
In recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been identified. Most of these receptors belong to one large family of proteins, all of which share homology with the protein import receptor importin beta (also named karyopherin beta). Members of this family have been classified as importins or exportins on the basis of the direction they carry their cargo. To date, the family includes 14 members in the yeast Saccharomyces cerevisiae and at least 22 members in humans. Importins and exportins are regulated by the small GTPase Ran, which is thought to be highly enriched in the nucleus in its GTP-bound form. Importins recognize their substrates in the cytoplasm and transport them through nuclear pores into the nucleus. In the nucleoplasm, RanGTP binds to importins, inducing the release of import cargoes. In ...
Structural biology and regulation of protein import into the nucleus
Journal of molecular biology, 2015
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,...
Importin α: a multipurpose nuclear-transport receptor
Trends in Cell Biology, 2004
The importin a/b heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin a binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin b, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin a back to the cytoplasm. In this article, we discuss control mechanisms that importin a exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin a genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin a that seem to be distinct from its housekeeping functions in nuclear transport.
PLoS ONE, 2013
The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-a (IMPa) adaptor protein, which in turn binds to IMPb to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPa in complex with the NLS of Prp20. We observe 1,091 Å 2 of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPa to contact bipartite NLS clusters 17 RAKKMSK 23 and 3 KR 4 , respectively. One notable feature of the major site is the insertion of Prp20NLS Ala 18 between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPa and cargo.
Evidence for distinct substrate specificities of importin α family members in nuclear protein import
1999
Importin ␣ plays a pivotal role in the classical nuclear protein import pathway. Importin ␣ shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin -dependent import pathway. In contrast to what is found for importin , several isoforms of importin ␣, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin ␣7. To analyze specific functions of the distinct importin ␣ proteins, we recombinantly expressed and purified five human importin ␣'s along with importin ␣ from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin ␣ proteins from humans or Xenopus bind their import receptor (importin ) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin ␣ proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin ␣3, whereas most of the other substrates were imported by all importin ␣ proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin ␣ proteins were revealed when two substrates were offered simultaneously.
Importin contains a COOH-terminal nucleoporin binding region important for nuclear transport
Journal of Cell Biology, 2003
roteins containing a classical NLS are transported into the nucleus by the import receptor importin  , which binds to cargoes via the adaptor importin ␣ . The import complex is translocated through the nuclear pore complex by interactions of importin  with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH 2 -terminal half of importin  . Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-P terminal region for nucleoporins is dramatically weaker than that of the NH 2 -terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin  to a similar extent ( ف 50%). An importin  mutant with a combination of mutations in the NH 2 -and COOH-terminal regions is completely inactive for nuclear import. Thus, importin  possesses two nucleoporin binding sites, both of which are important for its nuclear import function.
Journal of Cell Biology, 2002
he nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cyto-T plasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin ␣ /  -or transportin-dependent import.
Classical nuclear localization signals: definition, function, and interaction with importin α
Journal of Biological …, 2007
The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the -karyopherin importin , which mediates interactions with the nuclear pore complex, and the adaptor protein importin ␣, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.
Molecular and cellular biology, 1999
Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import ...