Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Nature ; 555(7697): 475-482, 2018 03 22.
Article in English | MEDLINE | ID: mdl-29539637

ABSTRACT

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.


Subject(s)
Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/chemistry , Nuclear Pore/metabolism , Saccharomyces cerevisiae/chemistry , Cross-Linking Reagents/chemistry , Mass Spectrometry , Models, Molecular , Protein Stability , Protein Transport , RNA Transport
2.
J Cell Biol ; 215(1): 57-76, 2016 Oct 10.
Article in English | MEDLINE | ID: mdl-27697925

ABSTRACT

Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30-60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC.


Subject(s)
Nuclear Pore Complex Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Biological Transport , Computer Simulation , Diffusion , Fluorescence Recovery After Photobleaching , Kinetics , Macromolecular Substances/metabolism , Molecular Weight , Mutation/genetics , Nuclear Pore/metabolism , Permeability , Protein Domains , Substrate Specificity , Thermodynamics , Time Factors
3.
Mol Cell Proteomics ; 11(5): 31-46, 2012 May.
Article in English | MEDLINE | ID: mdl-22357553

ABSTRACT

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a ~50 MDa complex consisting of ~30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa must be aided across by transport factors, most of which belong to a related family of proteins termed karyopherins (Kaps). These transport factors bind to the disordered phenylalanine-glycine (FG) repeat domains in a family of NPC proteins termed FG nups, and this specific binding allows the transport factors to cross the NPC. However, we still know little in terms of the molecular and kinetic details regarding how this binding translates to selective passage of transport factors across the NPC. Here we show that the specific interactions between Kaps and FG nups are strongly modulated by the presence of a cellular milieu whose proteins appear to act as very weak competitors that nevertheless collectively can reduce Kap/FG nup affinities by several orders of magnitude. Without such modulation, the avidities between Kaps and FG nups measured in vitro are too tight to be compatible with the rapid transport kinetics observed in vivo. We modeled the multivalent interactions between the disordered repeat binding sites in the FG nups and multiple cognate binding sites on Kap, showing that they should indeed be sensitive to even weakly binding competitors; the introduction of such competition reduces the availability of these binding sites, dramatically lowering the avidity of their specific interactions and allowing rapid nuclear transport.


Subject(s)
Active Transport, Cell Nucleus , Membrane Transport Proteins/chemistry , Nuclear Pore Complex Proteins/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae , beta Karyopherins/chemistry , Algorithms , Amino Acid Sequence , Bacterial Proteins/chemistry , Binding, Competitive , Escherichia coli , Models, Molecular , Protein Binding , Recombinant Proteins/chemistry , Serum Albumin, Bovine/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...