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Transcript of 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