nuclear structure nuclear pore import/ export signals receptors ran (directionality) fg platform...

Nuclear structureNuclear poreImport/ Export SignalsReceptorsRan (Directionality)FG Platform (Gating)Regulation

Nuclear Import & Export Overview

Origin Relates to TopologyParts

chromatinmatrixnucleolilamina (M-phase disassembly)poresinner membraneouter membranelumen (peri-nuclear space)

Nuclear Structure

Nuclear Lamina

EM of lamina

Mitotic Disassembly

Nuclear Pores

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

Figure 11-28

The nuclear pore complex

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/poreAlso 100 histones/min/pore etc.

Nuclear Cargo

125MD octagonal symmetry, 3-4K pores/cellcentral plugnuclear basketcytoplasmic fibers

About 30 (?) proteins “Nucleoporins” (FG repeats)Passive diffusion, limit 40-60KDChannel 9nm x 15 nm longOpens w/ signal: gating mech. unknownBidirectional

Nuclear Pore

9nm(40-60kDa)

40nm(up to 50MDa)

Size Exlcusion Limits

Model for passage of mRNPs through nuclear pore complexes

Not cleavedFolded proteins transportedNLS (nuclear localization signal)

Defined by transfection/microinjection experimentse.g. stretch of basic residues SV40 T=PKKKRKVdrives import of pyruvate kinase

NES (nuclear export signal)Defined by heterokaryon expte.g. leucine rich

Signals

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

3 TypesImportin beta (classic)NTF2 (for Ran import)TAP/p15 (mRNA export)

Identified with affinity chromatographypermeabilized cell assays

Importin beta (karyopherins)Large family of related Import & Export ReceptorsBind signals (direct or indirect), porins & RanGTP

cargo binding controlled by Randestabilizes import receptorsstabilizes export receptors

Receptors

Cytosol lostNPCs intact

Permeabilized Cells

Nuclear transport requires cytosolic factors and ATP

Importin beta structure

RCC1: stabilizes exchange intermediateRanGAP1, RanBP1, RanBP2: >105 fold cooperative

RanBP1 binds Ran->dissoc. then RanGAPRanGAP sumo/ RanBP2 complex on pore

NTF2: import receptor for RanGDP

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.

Transport cycles of exportins and importins use the chemical potential of a primary gradient, namely thatof RanGTP, to drive the directed transport of cargo molecules

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

Figure 11-34

CalineurinMasks NES

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

11.4 HIV Rev protein regulates the transport of unspliced viral mRNAs

Figure 11-38