#4 Ran GTPases

Nuclear pore complex

120MDa embedded complex in nuclear membrane, act as diffusion barrier and selective transport channel, composed of nucleoporins

Nuceloporins (Nups)

family of proteins that form NPCs, scaffold or FG repeat domains

Scaffold Nups

form ring complexes through membrane, anchor NPC, stabilize membrane curvature, attachment sites for FG

Inner ring complex - Nup53 complex

Outer, inner rings - Nup107-160 complex

FG Nups

Made of Phe/Gly repeat domains, binding sites fro nuclear transport receptors, defines the permeability characteristics of the NPC

Cytoplasmic filaments

extend from the NPC into cytoplasm, made of Nup358/RanBP2

NLS

Nuclear Localization signal (NLS): specific aa sequences, localize proteins to the nucleus

NES

Nuclear export signal (NES): specific aa sequences, export proteins from the nucleus

Nuclear transport receptors

recognize transport signals, importins/exportins, mediate transport through NPCs via FG repeat interaction

Ran GTPase system

generates asymmetry in RanGTP distribution, 200X more in nucleus

Ran

Ras-related nuclear protein (Ran): GTPase, binds guanine nucleotides, very stable (does not convert GTP without effectors)

RanGAP

Ran GTPase Activating Protein, causes Ran to convert GTP to GDP (hydrolizes triphosphate to diphosphate)

RanGEF

Ran Guanyl Nucleotide Exchange Factor, causes RanGDP to exchange GDP for GTP

Ran GTP Transport Receptors

superfamily, shuttle continuously between nucleus/cytoplasm

Importins

RanGTP transport receptor, high affinity for RanGTP, traffics RanGTP from cytoplasm into nucleus, RanGTP binding dissociates the import substrate

Exportins

RanGTP transport receptor, low affinity for RanGTP, cooperative binding with RanGTP and export substrate to allow export of RanGTP, GTP conversion causes dissociation and exportin returns to nucleus

Facillitated diffusion

requirs molecular binding event (importin/exportin bind FG nups) saturable due to limited binding sites

Selective phase model

Nuclear transport receptors dissolve FG Nups "mesh" by pulling apart FG-FG interactions, allowing translocation via NPC

Ran Switch loops

part of Ran structure conformational changes during GTP -> GDP

Loaded spring mechanism

switch 1/2 regions bring alpha-phosphate via Thr/Gly residues, upon GTP -> GDP hydrolysis, switch regions relax

Switch 3

in Ran, carboxyl-terminal tail packed against core of Ran during GDP binding, displaced when GTP binds

Importin B

RanGTP-binding transport receptor, composed of 19 helical HEAT repeats that form a spiral, contains IBB domain,  aa terminal arches align with switch regions of Ran, prevents enzymatic activity of Ran, multiple FG-repeat binding sites on surface

IBB Domain (importin B)

~35aa long N-terminal domain of Importin A that binds the C-terminal arch of Importin B

Exclusive binding (Importin B)

Ran-GTP and IBB-domain binding sites

CAS closed conformation

-RanGTP-exportin, requires contact of both RanGTP and cargo to form full exportin complex

-occurs when it is not bound, N-term and C-term arches interact, separation of this interaction required for open conformation

CAS opened confirmation

-RanGTP-exportin, requires contact of both RanGTP and cargo to form full exportin complex

-occurs when RanGTP binds due to induction/stabilization "

CRM1

exportin for NES cargo, donut shaped causing N-term and C-term HEAT repeats to interact, acidic loop interacts with Switch1 of Ran

-Ran: enclosed in donut, stabilizes ring closure, Switch 1 interacts with acidic loop of CRM1

-NES cargo: binds outside of donut, long-range allosteric communication with binding sites of cargo and RanGTP

RanBP family

-Ran-binding protein family, effector group 2, cause nuclear transport receptor/RanGTP complexes to dissociate to allow for RanGAP attack via their Ran binding domains

-Ran binding domains (RanBD): enwrapped by switch3 domain of Ran, causes the destabilization of the RanGTP-NTR interaction

RanBP1

cytoplasmic, contains NES to maintain localization

RanBP2

part of filaments of NPC

RanGAP

cytoplasmic, stimulate intrinsic GTPase activity of Ran, required for Ran to reach transition state of conformation, contains leucine-rich repeat domain attached to NPC filaments

RanGAP transition state

-conformation formed during GTP hydrolyzation (transferring gamma-phosphate to water), requires Q-residue of Ran that must be stabilized by RanGAP

-Asn131: aa of RanGap that helps orient the catalytic residue Gln69 of Ran

-Tyr39: aa of Ran, forms hydrogen bonds with beta-phosphate and Gln60 side chain of Ran to further support transition state stabilization

NTF2

nuclear transport factor 2, 15kDadimeric protein, transport receptor, recycles RanGDP to nucleus, can bind 2 RanGDP molecules via their switch II regions

RanGEF

(RCC1), ~45kDA, accelerate GDP/GTP exchange by acting as catalyst , disrupt nucleotide binding site and stabilization of nucleotide free form

RanGEF Disruption of binding site

Catalytic kick-out model: B-wedge of RCC1 pushes aside Ploop needed for nucleotide-binding

RanGEF Stabilization

-RanGEF binds Ran and stabilizes the complex of Ran by itself without GDP or GTP binding

-GTP binds preferably due to much higher concentration of GTP in nucleus