Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP 1 1Edited by I. B. Holland

Bayliss, Richard; Ribbeck, Katharina; Akin, Debra; Kent, Helen M.; Feldherr, Carl M.; Görlich, Dirk; Stewart, Murray

doi:10.1006/jmbi.1999.3166

Cited by 180 publications

(222 citation statements)

References 71 publications

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“…Indeed, in both the TIP4P-D and TIP4P-Ew simulations, the FSFG motifs formed multivalent contacts at many different sites (SI Appendix, Fig. S5A), including at previously experimentally determined sites (13,34) and sites suggested from simulations (33). In particular, we observed that a large number of interactions formed at the hydrophobic groove that bridges the two monomers of NTF2 and contains (but is not restricted to) the crystallographic binding site.…”

Section: Resultsmentioning

confidence: 57%

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Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Raveh

Karp

Sparks

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

131

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Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.nuclear pore | molecular dynamics | nucleoporins | transport factors | NMR T he nuclear pore complex (NPC) regulates macromolecular transport between the nucleus and the cytoplasm in all eukaryotic cells (1, 2). The NPC allows for passive diffusion of small molecules including sugars, ions, and water, while simultaneously imposing size-dependent exclusion of macromolecules without nuclear transport factor (TF) binding sites, generating unique nuclear and cytoplasmic compartments (3, 4). Larger macromolecules can bypass the selectivity barrier of the NPC by interacting with TFs that shuttle cargo through the central channel of the pore. In pathological conditions, including many cancers and viral infections, the NPC is hijacked and used to transport undesired particles, or it is modified to attenuate the cellular responses to disease onset (5, 6).TFs traverse the NPC by selective and reversible association with disordered phenylalanine-glycine (FG) repeat domains of the FG nucleoporin proteins (FG Nups), which line the surface of the NPC (7-14). Each FG repeat domain consists of 5-50 FG repeats. In turn, each FG repeat consists of a single FG motif of various sequence flavors, such as the FSFG and GLFG motifs, and 10-30 spacer residues that separate consecutive FG motifs (15, 16). The spacer residues are strongly hydrophilic (17) and rich ...

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Section: Resultsmentioning

confidence: 57%

“…TFs traverse the NPC by selective and reversible association with disordered phenylalanine-glycine (FG) repeat domains of the FG nucleoporin proteins (FG Nups), which line the surface of the NPC (7)(8)(9)(10)(11)(12)(13)(14). Each FG repeat domain consists of 5-50 FG repeats.…”

mentioning

confidence: 99%

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Raveh

Karp

Sparks

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

131

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“…Recent in vitro binding studies indicate that the interaction between NTF2 and nucleoporins is relatively weak (35,36). This suggests a model in which NTF2 transiently interacts with nucleoporins enabling the NTF2-RanGDP complex to move through the pore by hopping from one repeat to another (37).…”

mentioning

confidence: 99%

“…Bayliss et al (35) utilized the crystal structure of NTF2 bound to RanGDP to predict which residues in NTF2 might be involved in binding to nucleoporins. They identified residue Trp-7 as a potential site for FxFG binding and engineered a mutation in the rat NTF2 protein, W7A NTF2, with a reduced affinity for FxFG nucleoporins.…”

mentioning

confidence: 99%

Functional Analysis of the Hydrophobic Patch on Nuclear Transport Factor 2 Involved in Interactions with the Nuclear Porein Vivo

Quimby¹,

Leung²,

Bayliss³

et al. 2001

Journal of Biological Chemistry

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Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus.Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), 1 large proteinaceous channels that perforate the nuclear membrane. Protein cargoes to be imported into the nucleus contain internal sequences, termed nuclear localization signals (NLS), that target them to the nucleus (1-3). However, cargo does not interact directly with the NPC but is transported bound to soluble transport receptors of the importin-␤ family of proteins (4, 5). The recent purification and analysis of the yeast NPC indicates that the majority of the ϳ40 proteins that make up the NPC, collectively termed nucleoporins, are localized symmetrically throughout the pore complex (6). A subset of these proteins contains a phenylalanine-glycine (FG) repeat motif. These proteins appear to line the nuclear pore channel and can be subdivided further into either GLFG repeat-containing proteins or FxFG proteins (6,7). Although the precise mechanism by which the transport receptor-cargo complex translocates through the NPC is largely unknown, importin-␤ family transport receptors have been shown to interact with both the GLFG repeat and the FxFG repeat nucleoporins (8 -16) suggesting that these proteins are involved directly in the translocation process.The small GTPase Ran is central to nucleocytoplasmic transport. Although Ran is localized throughout the cell, ϳ80% is concentrated within the nucleus (17). The Ran guanine nucleotide exchange factor is tethered to DNA in the nucleus (18,19), suggesting that the majority of nuclear Ran is GTP-bound. On the other hand, RanGAP (Ran GTPase-activating protein) is predominantly cytoplasmic (20 -22), suggesting that the majority of cytoplasmic Ran is GDP-bound. Thus, the nucleotide bound state of Ran may act as a cellular marker that allows the transport machinery to distinguish between the nuclear and cytoplasmic compartme...

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“…The FGdomains can be further classified by their FG-repeat motifs, such as GLFG, FxFG and FG, although several FG-domains comprise more than one type of repeat (summarized in Table 1). Cargo selection relies on biochemical binding interactions between Kaps and the FG-repeat motifs (Radu et al, 1995;Bayliss et al, 1999Bayliss et al, , 2000Bayliss et al, , 2002aCushman et al, 2006). In doing so, Kap-FG binding is thought to somehow cause a transient breach or opening in the NPC barrier to make space for translocation to proceed.…”

Section: The Npc Paradoxmentioning

confidence: 99%

Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

Peleg

Lim

2010

Biological Chemistry

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Several biological mechanisms involve proteins or proteinaceous components that are intrinsically disordered. A case in point pertains to the nuclear pore complex (NPC), which regulates molecular transport between the nucleus and the cytoplasm. NPC functionality is dependent on unfolded domains rich in Phe-Gly (FG) repeats (i.e., FG-domains) that collectively act to promote or hinder cargo translocation. To a large extent, our understanding of FG-domain behavior is limited to in vitro investigations given the difficulty to resolve them directly in the NPC. Nevertheless, recent findings indicate a collective convergence towards rationalizing FG-domain function. This review aims to glean further insight into this fascinating problem by taking an objective look at the boundary conditions and contextual details underpinning FG-domain behavior in the NPC. Here, we treat the FG-domains as being commensurate with polymeric chains to address ambiguities such as for instance, how FG-domains tethered to the central channel of the NPC would behave differently as compared with their free-floating counterparts in solution. By bringing such fundamental questions to the fore, this review seeks to illuminate the importance of how such parameters can hold influence over the structure-function relation of intrinsically disordered proteins in the NPC and beyond.

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Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP 1 1Edited by I. B. Holland

Cited by 180 publications

References 71 publications

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Functional Analysis of the Hydrophobic Patch on Nuclear Transport Factor 2 Involved in Interactions with the Nuclear Porein Vivo

Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

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