1. Part 1: Intracellular trafficking Week 1: Transport through the nuclear pore complex Week 2: RNA transport, maturation & localization Week 3: ER translocation and vesicular transport 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 K128N SV40 T Ag contains small nuclear localization signal (NLS 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 + energyFraction A

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

11. The NLS receptor importin 

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

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 cargoCse1 without cargo

17. Structure of the exportin Cse1 with RanGTP and cargo

19. Establishment of a “Ran-gradient”

20. Ran cycle is coupled to transport cycle

21. Consumption of one GTP per transport cycle

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

23. Import-Export Cycle: the role of adaptors

24. Ran gradient throughout the cell cycle

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

26. THE METAZOAN NPC

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

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

30. Organization of the NPC

31. Brownian GateSelective Phase‘Oily Spaghetti Models for NPC function

32. 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