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Part 1: Intracellular trafficking

Part 1: Intracellular trafficking. Week 2: RNA transport, maturation & localization. Week 3: ER translocation and vesicular transport. Week 1: Transport through the nuclear pore complex. Week 4: Secretion & cell polarity.

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Part 1: Intracellular trafficking

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  1. Part 1: Intracellular trafficking Week 2: RNA transport, maturation & localization Week 3: ER translocation and vesicular transport Week 1: Transport through the nuclear pore complex Week 4: Secretion & cell polarity Week 5: Transport into other organelles (mitochondria, peroxisomes)

  2. Part 1: Intracellular trafficking Week 1: Transport through the nuclear pore complex Experimental approaches Signals and pathways The nuclear pore complex Soluble transport factors: import + export The functions of the small GTPase Ran Mechanism of translocation

  3. High concentration of nuclear pore complexes (NPCs) on nuclear envelopes from Xenopus oocytes

  4. Signals are necessary and sufficient for nuclear import

  5. Npl-coated gold particles can translocate across NPCs

  6. SV40 T Ag contains small nuclear localization signal (NLS) SV40 T Ag contains small nuclear localization signal (NLS SV40 T Ag K128N SV40 T Ag WT

  7. In vitro transport assay to nuclear import I

  8. In vitro transport assay to nuclear import II

  9. In vitro transport assay to nuclear import III Extract + energy Fraction A

  10. RanGTP displaces importin  from importin  importin  importin 

  11. The NLS receptor importin 

  12. The NLS receptor importin 

  13. The import receptor importin  Importin  Ran-GTP cargo NPC

  14. RanGTP displaces import cargoes from importins

  15. SUMMARY I. Signals target proteins into and out of the nucleus a. active transport b. signals are necessary and sufficient II.Transport occurs through the nuclear pore complex (NPC) a. NPC has aqueous channel b. no unfolding of cargoes is required c. transport is bi-directional III. Signals are recognized by soluble receptors: importins a. bind specific classes of cargo b. shuttle between cytoplasm and nucleus c. interact with nucleoporins

  16. Conformational changes that regulate cargo binding Cse1 with cargo Cse1 without cargo

  17. Structure of the exportin Cse1 with RanGTP and cargo

  18. Establishment of a “Ran-gradient”

  19. Ran cycle is coupled to transport cycle

  20. Consumption of one GTP per transport cycle

  21. Inversion of the Ran gradient leads to an inversion of the direction of transport nuclear RanGTP cytoplasmic RanGTP export cargo + exportin1

  22. Import-Export Cycle: the role of adaptors

  23. Ran gradient throughout the cell cycle

  24. S Nucleocytoplasmic transport G2 G1 Replication licensing RanGTPase Chromatin condensation Mitotic Spindle Assembly Nuclear Pore Assembly Kinetochore Assembly Nuclear Envelope Fusion M

  25. THE METAZOAN NPC

  26. THE YEAST NPC Rout et al.(2000) JCB 148:635

  27. Interaction between nuclear transport receptors and the FG-repeats within the NPC

  28. Organization of the NPC

  29. Models for NPC function Brownian Gate Selective Phase ‘Oily Spaghetti

  30. SUMMARY I. Short signals target proteins into and out of the nucleus Many proteins shuttle: regulation II. Many transport events are mediated by the importin b superfamily of importins and exportins III. Directionality is conferred by nuclear RanGTP Ran-GTP mediates substrate release (importins) or substrate binding (exportins) per import/export cycle ~ only one GTP is hydrolyzed RanGTP gradient provides energy for transport IV. NPC functions through facilitated diffusion FG rich repeats provide docking sites for transport factors

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