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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. 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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  1. Nuclear Import & Export Overview Nuclear structure Nuclear pore Import/ Export Signals Receptors Ran (Directionality) FG Platform (Gating) Regulation

  2. Nuclear Structure Origin Relates to Topology Parts chromatin matrix nucleoli lamina (M-phase disassembly) pores inner membrane outer membrane lumen (peri-nuclear space)

  3. Nuclear Lamina

  4. EM of lamina

  5. Mitotic Disassembly

  6. Nuclear Pores

  7. 11.4 Signal-mediated transport through nuclear pore complexes (NPC) The nuclear pore complex Figure 11-28

  8. 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.

  9. 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

  10. Size Exlcusion Limits 40nm (up to 50MDa) 9nm (40-60kDa)

  11. Model for passage of mRNPs through nuclear pore complexes

  12. 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

  13. 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

  14. 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

  15. Permeabilized Cells Cytosol lost NPCs intact

  16. Nuclear transport requires cytosolic factors and ATP

  17. Importin beta structure

  18. 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

  19. 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.

  20. FG Platform

  21. 11.4 A model for hnRNP-mediated export of mRNAs from the nucleus Figure 11-34

  22. Calineurin Masks NES

  23. 11.4 HIV Rev protein regulates the transport of unspliced viral mRNAs Figure 11-38

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