Saturday, 22 December 2012

Nuclear transport

Molecular mechanism of the nuclear protein iomport cycle by Stewart

Classic nuclear protein import cycle
Transport rate is 100- 1000 gargoes per minute per NPC. Driven by protein-protein interactions.  Cargo proteins with NLS are imported by carrier protein importin β, which binds them though adaptor protein importin α.  

In cytoplasm, cargo proteins with a classic NLS form an import complex with importin &alphalβ heterodimer. This facilitates movement through NPCs.

In nucleus, RanGTP binds importin-β.This dissociates import complex and releases cargo.  Importin β complexesd with RAnGTP is recycled to cytoplasm, Importin α is exported complexed with CAS and RanGTP.

Cytoplasmic RanGAP (Ran GATPase activating protein) stimulates Ran GTPase. This generates Ran GDP which dissociates from importins and relase them for anothe timport cycle.

Ran cycles between GTP and GDP bound states. They regulate interactions between cargoes and carriers.

Ran state is controlled by RanGEF which catalyses recharging of RAnGDP with RanGTP, and RanGAP which stimulates GTP hydrolysis.

RanGEF is nuclear. RAngAP is cytoplasmic. So nuclear Ran is in GTP bound state. Gyutoplasmic Ran is GDP bound.

RanGTP is exported from nucleus bound to β-karyopherin carriers. After RanGAP mediated GTP hydrolysis, cytoplasmic RanGDP is transported to nucleus by its specific carrier, nuclear transport factor-2 (NTF-2). It is recharged with GTP.

Conformation of key loops in Ran changes with nt state. This determines how importin β family transport factors interact with cargoes. Ran GTPase provides energy for nuclear transport.

4 steps: 1) assembly of cargo-import complex in cytoplasm 2) translocation through NPCs, 3) import-complex disassembly in nucleus, 4) importin recycling.

Step 1: import-complex assembly
Classic NLSs have 1 or 2 clusters of basic residues.  Molecular recognition of NLs is crucial for formation of important comple. It is mediated by spec sites on importin α. NLS-binding sites are formed from residues in a groove on inner concave surface.  Basic side chains in NLS interact spec with residues on inner surface of importin α.

N terminus of importin α binds importin-β through importin-β binding domain (IBB). It also facilitates cargo release in nucleus. IBB has cluster of basic residues similar to NLS. It can bind NLS-binding sites.

IBB connects importin α to importin β. It also has autoinhibitory role. When ti is not bound to importin β it comepets with NLSs for binding importin α. This conjtributes to cargo release.

Affinity of importin α that lack IBB domain for NLSs is higher than of full length protein. Importin α usually has higher affinity for NLSs than for IBB. Cargoes still bind full length importin α but with lower affinity than for IBB construct. 

Step 2: translocation through NPCs
Macromolecules larger than 40kS are excluded from NPCs. Only those bound to carriers can move through central transport channel. Smaller molecules diffuse through MPCs. Single molecule fluorescence studies indicate that movement of cargo-carrier complexes through NPCs is bidirectional and rapid.

Transport is not imposed by pore but by RanGTP-induced dissocation of cargo-carrier import complex in nucleus.

FG nucleoporins are a subset of NPC proteins with FG sequence repeats. May help mediate movement of cargo-carrier complexes through NPCs and exclude other molecules from central transport channel.  Phe side chains of hydrophobic FG repeat core bind cavities on surface of carriers. The interaction is weal. It is transient enough to enable rapid transport of cargo-carrier complexes.

Models of selective transport. FG repeat concentration in NPC transport channel is high. This may obstruct passage of macromolecules. Molecular crowging may exclude them rntropically. The FG chains get in each other's way. Chain is arranged in ordered manner and have lower entropy.

Alternately, FG repeats may form a sievelike gel through interactions between FG cores. Diffusion of particles in crosslinked gel depends on gel's pore size.

Cargo-carrier complexes are proposed to overcome barrier by FG nucleoporins by interacting with FR repeat cores. Molecular crowind model suggest enthalpy if binding compensates for entropic penalty of penetrating closely packed nucleoporin chains. Hydrogel/sieve model proposes that interaction with carrier disrupts interactions between FG repeat cores that generate gel. This transiently opens adjacent meshes in gel.

FG repeats could influence transport rates by concentraing components of classic nuclear protein import machinery.

Step 3: import-complex disassembly
Nuclear RanGTP dissociates cargo-carrier import complex. It imposes directionality on transport. Rapid diffusion of complex back and forth in channel maintains equilibrium across NPC. Removing complex from nuclear side by dissociation by RanGTP causes net flow of cargo-carrier complex from cytoplasm to nucleus. This restores equilibrium.

Thermal energy (Brownian motion) allows cargo bound to carrier diffuse rapidly in either direction but cargo cannot return to cytoplasm after it dissociated from its carrier in nucleus. RanGTP hydrolysis energy rectifies Brownian motion of cargo-carrier complex. Generates a thermal ratchet that imposes directionality on transport by dissociating import complex.

a | Structure of the Saccharomyces cerevisiae importin-beta:RanGTP74 complex showing how importin-beta (shown in yellow) coils around Ran (blue). Bound GTP is shown in cyan, whereas the Ran switch I loop is in red and the switch II loop is in green. b | Schematic representation of the structure shown in part a. The HEAT repeats from which importin-beta is constructed are shown as yellow blocks (H1–19). The residues that form the interface between importin-beta (yellow) and RanGTP (blue) are indicated (Ran switch I residues are in red and switch II are in green)7. RanGTP binds at three different sites. The first site primarily involves residues in the switch II loop (green) that bind to HEAT repeats 1–4; the second site involves a basic patch on Ran (K134, H139, R140 and K141) that binds electrostatically to acidic residues in HEAT repeats 7 and 8; and the third site primarily involves residues in the switch I loop (red) that interact with HEAT repeats 12–15. The third site is crucial for locking importin-beta into a conformation in which it cannot bind the importin-beta binding (IBB) domain of importin-alphac | Two views rotated by 90° about the vertical that illustrate the conformational changes in importin-beta that take place when RanGTP binds to importin-beta, dissociating the importin-alpha:beta complex. The cyan trace shows the conformation of importin-beta when bound to the IBB domain (magenta trace) in the absence of RanGTP. The yellow trace shows the conformation of importin-beta when bound to RanGTP. The dramatic conformational change that follows Ran binding disrupts the precise match between the importin-beta helicoid and the IBB alpha-helix75. See also Supplementary information S4 (movie). Parts a and b of the figure are reproduced

Dissociation of importin αβ complex. Binding of RanGTP to importin β dissociates importin αβ complex. This releases cargo. RanGTP releases importin α IBB domain by inducing conformational change in importin β.

Cargo release. When released from importin β by RanGTP, IBB domain competes with NLS of cargo for binding to importin α. This decreases affinity of cargo for importin α. facilitates release.

Step 4: importin recycling
After disassembly by RanGTP, importins must be recycled to cytoplasm. Importin β is recycled complexed with RanGTP. Importin α is exported actively by CAS.

CAS is structurally similar to importin β.  It is based on 19 HEAT repeats. Mutations interfering with binding of IBB to either NLS site or CAS prevent formation of export complex.

Export complex can only form wheh cargo has been released from importin α.

IBB must also diaplce Nup2/NUP50 bound to importin α.  This is difficult as ucleoporins bind importin α with higher affinity than NLSs.

Nup2/NUP50 may be removed. CAS displaces Nup2/NUP50 from high affinity site at importin α terminus. This facilitates IB domain disaplcing Nup2/NUP5- from NLS binding site.

When importin β-RanGTP and CAS-RanGTP-importin α compelxes return to cytoplasm through NPCs, cytoplasmic RanGDP (with RanBP1, which removes RanGTP from importin β) stimulates GTP hydrolysis. This releases Ran from importin β and CAS. Frees importins for another import cycle.

a | The structure of the Saccharomyces cerevisiae CAS:importin-alpha:RanGTP export complex33 shows how the importin-beta binding (IBB) domain (magenta) is locked against importin-alpha (green), occupying the same site as that bound by nuclear localization signals (NLSs) (see also Fig. 2e). This interaction is crucial for the formation of the complex and can only occur when cargo has been released. As observed with importin-beta, CAS (yellow) coils around both importin-alpha and RanGTP (cyan) in the complex. b | Illustration of the substantial conformational change between CAS in the export complex33 (yellow) and the isolated molecule79 that is formed after RanGTP hydrolysis in the cytoplasm (cyan) (see also Supplementary information S9S10 (movies)). c | Nup2 (red; and its metazoan counterpart, NUP50 (blue)) binding to the high-affinity site at the C terminus of importin-alpha would clash with CAS in the export complex. Therefore, CAS first displaces Nup2/NUP50 from the high-affinity site at the C terminus of importin-alpha, which then facilitates the IBB domain displacing it from the NLS-binding sites. This explains how CAS binding can displace Nup2/NUP50 (ref. 35). d | Schematic illustration of the series of interactions in S. cerevisiae involving Nup2 (red) and CAS (yellow) that leads to displacement of the NLS and generates a molecular ratchet to prevent futile cycles in which the cargo is returned to the cytoplasm35. After NLS is released from importin-beta by RanGTP (cyan), the IBB domain (magenta) can displace the NLS-containing cargo from importin-alpha, allowing CAS complexed to RanGTP to bind. This enables recycling of importin-alpha to the cytoplasm. This process is catalysed by Nup2 that first displaces the NLS and is then itself displaced by CAS. This series of interactions also generates a molecular ratchet to prevent futile transport cycles. Part a is modified with permission from Ref. 33 © (2004) Macmillan Magazines Ltd. Part b is modified from Ref. 35 © (2005) Macmillan Magazines Ltd.

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