Nuclear import export overview
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Nuclear Import & Export Overview. Nuclear structure Nuclear pore Import/ Export Signals Receptors Ran (Directionality) FG Platform (Gating) Regulation. Nuclear Structure. Origin Relates to Topology Parts chromatin matrix nucleoli lamina (M-phase disassembly) pores

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Nuclear Import & Export Overview

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Nuclear import export overview

Nuclear Import & Export Overview

Nuclear structure

Nuclear pore

Import/ Export Signals

Receptors

Ran (Directionality)

FG Platform (Gating)

Regulation


Nuclear structure

Nuclear Structure

Origin Relates to Topology

Parts

chromatin

matrix

nucleoli

lamina (M-phase disassembly)

pores

inner membrane

outer membrane

lumen (peri-nuclear space)


Nuclear lamina

Nuclear Lamina


Em of lamina

EM of lamina


Mitotic disassembly

Mitotic Disassembly


Nuclear pores

Nuclear Pores


11 4 signal mediated transport through nuclear pore complexes npc

11.4 Signal-mediated transport through nuclear pore complexes (NPC)

The nuclear pore complex

Figure 11-28


Nuclear cargo

Nuclear Cargo

  • Imported

  • Polymerases

  • Histones

  • Transcription factors

  • Ribosomal proteins

  • Exported

  • tRNAs

  • mRNPs

  • Ribosomal subunits

  • Transcription factors

106 ribos=>560K ribo proteins imported/min

14,000 ribo subs exported/min

3-4K pores/cell=> 150 ribo proteins/min/pore

Also 100 histones/min/pore etc.


Nuclear pore

Nuclear Pore

125MD octagonal symmetry, 3-4K pores/cell

central plug

nuclear basket

cytoplasmic fibers

About 30 (?) proteins “Nucleoporins” (FG repeats)

Passive diffusion, limit 40-60KD

Channel 9nm x 15 nm long

Opens w/ signal: gating mech. unknown

Bidirectional


Size exlcusion limits

Size Exlcusion Limits

40nm

(up to 50MDa)

9nm

(40-60kDa)


Model for passage of mrnps through nuclear pore complexes

Model for passage of mRNPs through nuclear pore complexes


Signals

Signals

Not cleaved

Folded proteins transported

NLS (nuclear localization signal)

Defined by transfection/microinjection experiments

e.g. stretch of basic residues SV40 T=PKKKRKV

drives import of pyruvate kinase

NES (nuclear export signal)

Defined by heterokaryon expt

e.g. leucine rich


Nuclear import export overview

11.4 Heterokaryon assay demonstrating that human hnRNP A1 protein (red) can cycle in and out of the cytoplasm but human hnRNP protein C (grn) cannot

Figure 11-32


Receptors

Receptors

3 Types

Importin beta (classic)

NTF2 (for Ran import)

TAP/p15 (mRNA export)

Identified with

affinity chromatography

permeabilized cell assays

Importin beta (karyopherins)

Large family of related Import & Export Receptors

Bind signals (direct or indirect), porins & RanGTP

cargo binding controlled by Ran

destabilizes import receptors

stabilizes export receptors


Permeabilized cells

Permeabilized Cells

Cytosol lost

NPCs intact


Nuclear transport requires cytosolic factors and atp

Nuclear transport requires cytosolic factors and ATP


Importin beta structure

Importin beta structure


Nuclear import export overview

RCC1: stabilizes exchange intermediate

RanGAP1, RanBP1, RanBP2: >105 fold cooperative

RanBP1 binds Ran->dissoc. then RanGAP

RanGAP sumo/ RanBP2 complex on pore

NTF2: import receptor for RanGDP


Nuclear import export overview

Facilitated Diffusion

Transport cycles of exportins and importins use the chemical potential of a primary gradient, namely that

of RanGTP, to drive the directed transport of cargo molecules

On its own, translocation per se would be a fully reversible process that would only allow an equilibration of cargoes between nucleus and cytoplasm. Directional transport is achieved by Ran-regulated cargo loading and unloading of the transport receptors. This also constitutes the input of energy in the form of one molecule of GTP hydrolyzed per transport cycle.


Nuclear import export overview

FG Platform


11 4 a model for hnrnp mediated export of mrnas from the nucleus

11.4 A model for hnRNP-mediated export of mRNAs from the nucleus

Figure 11-34


Nuclear import export overview

Calineurin

Masks NES


11 4 hiv rev protein regulates the transport of unspliced viral mrnas

11.4 HIV Rev protein regulates the transport of unspliced viral mRNAs

Figure 11-38


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