Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-β/karyopherin-β1; and (ii) the karyopherin-β2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-β2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-α•cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
a b s t r a c tChloride intracellular channel proteins (CLICs) are distinct from most ion channels in that they have both soluble and integral membrane forms. CLICs are highly conserved in chordates, with six vertebrate paralogues. CLIC-like proteins are found in other metazoans. CLICs form channels in artificial bilayers in a process favoured by oxidising conditions and low pH. They are structurally plastic, with CLIC1 adopting two distinct soluble conformations. Phylogenetic and structural data indicate that CLICs are likely to have enzymatic function. The physiological role of CLICs appears to be maintenance of intracellular membranes, which is associated with tubulogenesis but may involve other substructures.
The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.
It has been reported that a human chloride intracellular channel (CLIC) protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localization signal (NLS). The CLIC4 NLS adopts an α‐helical structure in the native CLIC4 fold. It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin‐α. In this study, we have determined the X‐ray crystal structure of a truncated form of importin‐α lacking the importin‐β binding domain, bound to a CLIC4 NLS peptide. The NLS peptide binds to the major binding site in an extended conformation similar to that observed for the classical simian virus 40 large T‐antigen NLS. A Tyr residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side‐chain hydrogen bonds to the importin‐α backbone. This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin‐α nuclear import pathway, provided that CLIC4 can undergo a conformational rearrangement that exposes the NLS in an extended conformation. Database Structural data are available in the protein Data Bank under the accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3OQS. Structured digital abstract http://www.uniprot.org/uniprot/Q9Y696 and http://www.uniprot.org/uniprot/P52293 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8118302
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