Gradient of Increasing Affinity of Importin β for Nucleoporins along the Pathway of Nuclear Import

Ben-Efraim, Iris; Gerace, Larry

doi:10.1083/jcb.152.2.411

Cited by 264 publications

(261 citation statements)

References 28 publications

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“…The Nup214-Nup88 Subcomplex Is Dispensable for Basic NES-mediated Nuclear export-The strong in vitro interaction between Nup214 and CRM1 (29 -31), suggests that this nucleoporin has an important stimulatory role in NES-mediated nuclear export (16,30,31). To test for this possibility, we depleted Nup214 by expression of Nup214-specific shRNAs in human cells and recorded the nucleocytoplasmic localization of NES-reporter proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Directionality of transport through the NPC is determined by the Ran GTP/GDP gradient, which exists between the nucleus and the cytoplasm (13)(14)(15), and possibly by the presence of specific high affinity binding sites for transport receptors located at either the nuclear or cytoplasmic faces of the NPC (16). Although the general NPC architecture is symmetric, the localization of several nucleoporins is restricted to either the nuclear or the cytoplasmic face of the NPC (3), supporting the idea that nucleocytoplasmic asymmetry might be established by asymmetric distribution of specific binding sites at the NPC.…”

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confidence: 99%

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Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/ hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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“…One of the controversies surrounding the field involves the origin of the 'paradoxical' selective gating mechanism/permeability barrier, which remains largely unknown. Based on strong indications that the barrier is formed by the FG-repeat domains, several attempts have been made to model and make sense of the plausible translocation mechanisms (Rout et al 2000;Ben-Efraim and Gerace 2001;Macara 2001;Ribbeck and Gorlich 2002). The Brownian affinity gate model (Rout et al 2000(Rout et al , 2003 proposes that the entropic behavior of peripheral FGrepeat nucleoporins acts as a substantial barrier to inert cargo.…”

Section: Nucleocytoplasmic Transport Across the Nuclear Pore Complex:mentioning

confidence: 99%

From the trap to the basket: getting to the bottom of the nuclear pore complex

2006

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Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the doublemembraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of highresolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.

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“…Hence, Kap-FG Nup binding is characterized by highly multivalent interactions, 26,27 which are recognized to enhance stability and specificity due to strong binding avidity. 28 As raised by Tetenbaum-Novatt et al, 29 the known sub-mM Kapb1-FG domain binding affinities 11,[30][31][32] may "ensure" NPC transport selectivity but contradict the rapid »5 ms dwell times of nucleocytoplasmic transport cargoes in vivo 33 and in vitro. [34][35][36] It is intriguing that even FG Nup-coated nanopores are able to recapitulate both the selectivity and speed of Kapb1 translocation with a dwell time of »2.5 ms, 37 In fact, the NPC problem conflates specific binding, translocation speed and nanoscale spatial constraints.…”

Section: Introductionmentioning

confidence: 99%