Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α (original) (raw)
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Journal of Molecular Biology, 2000
Importin-a is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-a, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-a. The nucleoplasmin NLS-importin-a complex shows, for the ®rst time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein. Abbreviations used: Arm repeats, armadillo repeats; Impa(70-529), importin-a lacking the N-terminal 69 residues; NLS, nuclear localization sequence; NPC, nuclear pore complex; SV40, simian virus 40; SV40 NLS, peptide 126 PKKKRKV 132 corresponding to the NLS of the SV40 large T-antigen; nucleoplasmin NLS, peptide 155 KRPAATKKAGQAKKKK 170 corresponding to the NLS of nucleoplasmin.
Nuclear retention of importin α coordinates cell fate through changes in gene expression
The EMBO Journal, 2011
Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin a in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin a performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin a2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin a2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin a2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.
Molecular and Cellular Biology, 2006
The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin alpha3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin alpha binding and transport.
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 ...
Journal of Biological Chemistry, 2010
Importin-␣ is the nuclear import receptor that recognizes the classic monopartite and bipartite nuclear localization sequences (cNLSs), which contain one or two clusters of basic amino acids, respectively. Different importin-␣ paralogs in a single organism are specific for distinct repertoires of cargos. Structural studies revealed that monopartite cNLSs and the C-terminal basic clusters of the bipartite cNLSs bind to the same site on importin-␣, termed the major cNLS-binding site. We used an oriented peptide library approach with five degenerate positions to probe the specificity of the major cNLS-binding site in importin-␣. We identified the sequences KKKRR, KKKRK, and KKRKK as the optimal sequences for binding to this site for mouse importin-␣2, human importin-␣1, and human importin-␣5, respectively. The crystal structure of mouse importin-␣2 with its optimal peptide confirmed the expected binding mode resembling the binding of simian virus 40 large tumor-antigen cNLS. Binding assays confirmed that the peptides containing these sequences bound to the corresponding proteins with low nanomolar affinities. Nuclear import assays showed that the sequences acted as functional cNLSs, with specificity for particular importin-␣s. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-␣; the results will contribute to understanding of the sequence determinants of cNLSs, and may help identify as yet unidentified cNLSs in novel proteins.
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.
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.