Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import

Stewart, Murray; Baker, Rosanna P.; Bayliss, Richard; Clayton, Lesley; Grant, Richard P.; Littlewood, Trevor D.; Matsuura, Yasushi

doi:10.1016/s0014-5793(01)02489-9

Cited by 102 publications

(81 citation statements)

References 61 publications

(135 reference statements)

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“…Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6,7). Release of the import complex from nuclear pore complex binding sites as well as the final release of the import cargo into the nucleoplasm is mediated by the small GTPase Ran, which binds to importin ␤ in its nuclear GTP-bound form (8).…”

mentioning

confidence: 99%

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

Chen

Ben-Efraim

Mitrousis

et al. 2005

Journal of Biological Chemistry

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Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin ␣/␤.Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257 GKISKH-WTGI 266 . This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin ␣. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin ␣, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin ␣.Nuclear transport is an active signal-mediated process that requires, in most cases, soluble transport factors and specific import signals. Two families of transport receptors have been identified in the importin ␤ superfamily, which is involved in both nuclear import and export, and the TAP superfamily that mediates nuclear export. Transport receptors recognize specific nuclear localization signals (NLSs) 1 and nuclear export signals exposed on the molecular surface of cargoes. In the classical nuclear import pathway proteins bearing a classical SV40-like NLS (PKKKRKV) are recognized by the importin ␣/␤ heterodimer (also known as karyopherin ␣/␤) (1-3). Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6, 7

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confidence: 99%

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

Chen

Ben-Efraim

Mitrousis

et al. 2005

Journal of Biological Chemistry

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“…Transport through the channel requires binding of protein or RNA cargoes to soluble transport receptors and is mediated by the phenylalanine-glycine (FG) repeats that characterize most nucleoporins. The two most common repeats that are found in nucleoporins and are often present in many copies along the whole molecule are based on GLFG or FXFG cores (1)(2)(3).…”

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confidence: 99%

The Mtr2-Mex67 NTF2-like Domain Complex

Senay

Ferrari

Rocher

et al. 2003

Journal of Biological Chemistry

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The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apoMtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.In eukaryotic cells, the nuclear and cytoplasmic compartmentalization requires that a large number of molecules be continuously transported through the nuclear pore complex (NPC), 1 a huge macromolecular structure that spans the nuclear envelope. Nucleoporins represent a subset of the NPC components and form a dense network of proteins that line the channel of the NPC. Transport through the channel requires binding of protein or RNA cargoes to soluble transport receptors and is mediated by the phenylalanine-glycine (FG) repeats that characterize most nucleoporins. The two most common repeats that are found in nucleoporins and are often present in many copies along the whole molecule are based on GLFG or FXFG cores (1-3).The best characterized pathway of protein import and export involves members of the conserved family of transport receptors called karyopherins/importin-␤, also known as exportins/ importins. The karyopherin transport factors share a common structural framework and respond to the small GTPase, Ran, to bind or release their cargo within the appropriate cellular compartments. Meanwhile, Ran must also shuttle across the NPC to equilibrate its nuclear level; this is performed via a specific nuclear import factor known as nuclear transport factor 2 (NTF2) (4 -8).Unlike this well established protein transport pathway, no karyopherin family member that would function in general mRNA export has been identified. Instead, recent studies in yeast and metazoans have pointed to several highly conserved proteins that are known as nuclear export factor (NXF) and are specifically required for mRNA export, but do not include karyopherin or NTF2 (9). Among members of the NXF family, the best characterized candidate is the Saccharomyces cerevisiae protein Mex67, whose conserved metazoan counterpart is known as TAP or NXF. Mex67 interacts with both bulk poly(Aϩ) RNA and nuclear p...

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“…Interacts In Vitro with Tf1-Gag, the Importin ␤ Kap95 and the Importin ␣'s Imp1p and Cut15 FG-Nups interact directly with a broad range of transport factors and there is compelling in vivo evidence that interaction between the carriers and FG-Nups is important for at least one stage of translocation through NPCs (reviewed in Ryan and Wente, 2000;Allen et al, 2001;Stewart et al, 2001). Studies have demonstrated interactions between FXFG or GLFG cores of nucleoporins and transport factors (Bayliss et al, 2000(Bayliss et al, , 2002a and also binding of transport factors to non-FG regions of nucleoporins (Pyhtila and Rexach, 2003).…”

Section: Nup124pmentioning

confidence: 99%

“…Both Cut15p and Imp1p were able to import classical NLS-containing substrates in vivo and in vitro into the nucleus, implying that each is an authentic importin ␣ with possibly common binding partners (Umeda et al, 2005). There is compelling in vivo evidence that interaction between importin ␣ and FG Nups may be the basis for the latter to either concentrate carrier-cargo complexes at the NPC entrance and/or participate in a sequence of docking and undocking interactions as the carrier-cargo complexes transit through NPCs (Stewart et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Multiple Conserved Domains of the Nucleoporin Nup124p and Its Orthologs Nup1p and Nup153 Are Critical for Nuclear Import and Activity of the Fission Yeast Tf1 Retrotransposon

Sistla

Wang

Balasundaram

2007

MBoC

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The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.

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Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import

Cited by 102 publications

References 61 publications

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

The Mtr2-Mex67 NTF2-like Domain Complex

Multiple Conserved Domains of the Nucleoporin Nup124p and Its Orthologs Nup1p and Nup153 Are Critical for Nuclear Import and Activity of the Fission Yeast Tf1 Retrotransposon

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