Summary of current models of npc gating during

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Summary of current models of NPC gating during karyopherin translocation- Low affinity Low affinity Hi affinity Hi affinity Medium affinity Medium affinity RanGTP affinity gradient model “oily-spaghetti” model selective phase model virtual gate model
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Virtual Gate/Polymer Brush Hydrogel Two Gate side view Current hypothetical models of NPC conduit architecture Key Questions: ! Do FG domains interact via hydrophobic FG motifs? ! Do FG domains prefer random or extended-coil configurations at the NPC as shown? ! Do FG nups form a quaternary structure at the NPC? FG domains as random-coils FG domains as extended-coils Virtual Gate/Polymer Brush non-cohesive FG motifs Selective Phase/Hydrogel cohesive FG motifs top view, facing cytosol
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Bead-immobilized GST-nups binding no binding soluble CFP-nups + Equilibrium-based, low-affinity protein interaction assay Do FG domains of nups form weak inter-molecular interactions?
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Do FG domains bind each other via FG motif interactions? Bead-immobilized GST-FG domain binding no binding Soluble CFP-FG domain + Use an equilibrium-based, low-affinity protein interaction assay (BEAD HALO) Affinity range to at least 100 ! M Patel et al & Rexach, Cell 2007 F > A mutant + Soluble CFP-Nup116 FG domain wild type Beads coated with a Nup116 FG domain GLFG motifs GLAG motifs
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A summary of the interactivity-profile between the disordered FG domains of Nups and their location at the NPC The Two Gate Model Nup159 Nup42 Nup116 Nup100 Nup57 Nup49 nNup145 Nup1 Nup60 Nup159 Nup42 Nup116 Nup100 Nup57 Nup49 nNup145 Nup1 Nup60 Nup2 Nup2 FG Nups with FxFG motifs and a high charge content FG Nup with triple FG motifs and a high charge content FG Nups with GLFG motifs and a low charge content Charge content governs FG domain interactivity
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The topology and hydrodynamic dimensions of all S. cerevisiae FG Nups Some FG domains may function as collapsed coil globules connected to the NPC scaffold via relaxed or extended-coils: the ‘stalks’ The stalks may act as springs, dynamic tethers, or bumpers Collapsed-coil, low charge-content, cohesive Relaxed or extended-coil, high charge content, non-cohesive Folded domain Collapsed-coil, high charge-content, non-cohesive NPC anchor domain Nup42 8 nm Nup49 5 nm Nup57 7 nm Nup116 10 & 8 nm Nup100 10 & 7 nm shrub tree Nup145N 5 & 6 nm - + + + - - Nup60 7 nm + - + - - + Nup159 10 & 13 nm Nup2 4 & 13 nm - - - - - - + - + - - + + Nsp1 6 & 12 nm Nup1 7 & 14 nm Hydrodynamic size (diameter) assuming a spherical shape - - + - + - + + - + + Total hydrodynamic volume occupied by collapsed-coil FG domains: Total volume occupied by relaxed-to-extended coil FG domains and stalks: Total volume occupied by FG domains and stalks: NPC conduit volume (~35 nm height x 28 nm radius): 20,471 nm 3 67,844 nm 3 88,315 nm 3 86,162 nm 3
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Building the Forest Model: The hydrodynamic volume occupied by disordered FG domains of Nups in a scale drawing of the NPC conduit At first glance it would seem that FG nups are not large enough to contact others anchored at opposite sides of the conduit; hence a central conduit would be always open… But a polymer-brush effect ( i.e. entropic exclusion by crowding) could force them to elongate
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