Permeating the nuclear pore complex

Kapon, Ruti; Naim, Bracha; Zbaida, David; Nevo, Reinat; Tsabari, Onie; Reich, Ziv

doi:10.4161/nucl.1.6.13112

Cited by 8 publications

(6 citation statements)

References 64 publications

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“…Indeed, NPC-contacting domains of transport receptors are generally presumed to be surface-located hydrophobic patches and are dependent on protein conformation (reviewed in Ref. 41). In this context we note that changes to a single residue (Tyr-654) of the R10 -12 sequence were sufficient to accelerate ␤-catenin import to a rate comparable with that of importin-␤ (Fig.…”

Section: Discussionmentioning

confidence: 93%

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Sharma

Jamieson

Johnson

et al. 2012

Journal of Biological Chemistry

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Background:The nuclear localization of ␤-catenin is directly linked to its cancer causing activity. Results: Armadillo repeats (10 -12) mediate nuclear transport of ␤-catenin through direct interaction with specific nuclear pore complex proteins. Conclusion: ␤-Catenin can function like a nuclear transport receptor in its ability to translocate independently through the nuclear pore complex. Significance: ␤-Catenin may transport specific binding partners between the nucleus and cytoplasm in response to Wnt signaling.␤-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. ␤-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10 -12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of ␤-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of ␤-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of ␤-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10 -12, and ␤-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of ␤-catenin to a greater extent than that of importin-␤. The Arm R10 -12 sequence facilitated transport even when ␤-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated ␤-catenin transport through direct interaction with the NPC.The canonical Wnt signaling pathway regulates normal tissue development and maintenance through its effector molecule, ␤-catenin. In the absence of Wnt signaling ␤-catenin accumulates at adherens junctions. ␤-Catenin levels are regulated through degradation by a destruction complex, comprising of adenomatous polyposis coli (APC), 4 axin, casein kinase I, and glycogen synthase kinase-3␤, which phosphorylates and subsequently ubiquitinates ␤-catenin at the N terminus, leading to its degradation by the proteosome (1, 2). On receiving Wnt signals or through misregulation of the degradation pathway, ␤-catenin is stabilized and accumulates in the nucleus where it binds to lymphoid enhancer factor-1 (LEF/ TCF) transcription factors and activates transcription of tumor promoting genes involved in cell migration...

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Section: Discussionmentioning

confidence: 93%

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Sharma

Jamieson

Johnson

et al. 2012

Journal of Biological Chemistry

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“…The results of several previous investigations have suggested that the surface hydrophobicity of large cargo proteins (>40 kDa) itself, independent of transport receptors, could enable successful nucleocytoplasmic transport of large cargos [45] – [47] . However, whether there is a distinct principle or a similar mechanism between cargo-surface-hydrophobicity-driven nuclear transport and transport-receptor-driven cargo translocation remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Role of Molecular Charge in Nucleocytoplasmic Transport

Goryaynov

Yang

2014

PLoS ONE

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Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.

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“…These NPCs allow passive diffusion of small solutes, as well as mediated selective transport of macromolecules in a signal‐dependent manner called active transport. The mechanism of active translocation through NPC involves interactions between the soluble carriers and proteins present inside the NPC (Rout et al, ; Conti et al, ; Minakhina et al, ; He et al, ; Kapon et al, ; Strambio‐De‐Castillia et al, ; Wente and Rout, ; Schuldt, ). The passive transport takes place by simple diffusion through the concentration gradient and the rate of diffusion will critically depend on the size of the pores.…”

Section: Introductionmentioning

confidence: 99%

Passive permeability and effective pore size of HeLa cell nuclear membranes

Samudram

Mangalassery

Kowshik

et al. 2016

Cell Biology International

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Nuclear pore complexes in the nuclear membrane act as the sole gateway of transport of molecules from the cytoplasm to the nucleus and vice versa. Studies on biomolecular transport through nuclear membranes provide vital data on the nuclear pore complexes. In this work, we use fluorescein isothiocyanate-labeled dextran molecules as a model system and study the passive nuclear import of biomolecules through nuclear pore complexes in digitonin-permeabilized HeLa cells. Experiments are carried out under transient conditions in the time lapse imaging scheme using an in-house constructed confocal laser scanning microscope. Transport rates of dextran molecules having molecular weights of 4-70 kDa corresponding to Stokes radius of 1.4-6 nm are determined. Analyzing the permeability of the nuclear membrane for different sizes the effective pore radius of HeLa cell nuclear membrane is determined to be 5.3 nm, much larger than the value reported earlier using proteins as probe molecules. The range of values reported for the nuclear pore radius suggest that they may not be rigid structures and it is quite probable that the effective pore size of nuclear pore complexes is critically dependent on the probe molecules and on the environmental factors.

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Permeating the nuclear pore complex

Cited by 8 publications

References 64 publications

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Role of Molecular Charge in Nucleocytoplasmic Transport

Passive permeability and effective pore size of HeLa cell nuclear membranes

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