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

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

3. Nuclear Lamina

4. EM of lamina

5. Mitotic Disassembly

6. Nuclear Pores

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

8. Imported Polymerases Histones Transcription factors Ribosomal proteins Exported tRNAs mRNPs Ribosomal subunits Transcription factors 10 6 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 Cargo

9. 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 Nuclear Pore

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

12. Model for passage of mRNPs through nuclear pore complexes

13. 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 Signals

15. 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

16. 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 Receptors

17. Cytosol lost NPCs intact Permeabilized Cells

18. Nuclear transport requires cytosolic factors and ATP

19. Importin beta structure

22. RCC1: stabilizes exchange intermediate RanGAP1, RanBP1, RanBP2: >10 5 fold cooperative RanBP1 binds Ran->dissoc. then RanGAP RanGAP sumo/ RanBP2 complex on pore NTF2: import receptor for RanGDP

23. 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 that of RanGTP, to drive the directed transport of cargo molecules

25. 11.4 A model for hnRNP-mediated export of mRNAs from the nucleus Figure 11-34

27. Calineurin Masks NES

29. 11.4 HIV Rev protein regulates the transport of unspliced viral mRNAs Figure 11-38