1. The Secretory Pathway - Classic Experiments - ER Translocation - Membrane budding and fusion

2. Intracellular Transport Pathways

4. Vesicular Transport

6. Pulse-chase: Palade and co-workers 3H-Leu pulse to monitor secretion in pancreatic cells 10’ Golgi45’ Secretory Granules

7. Modern version: GFP allows visualization of transport 1. ts VSV G accumulates in ER 2. After lowering T: ER exit sites 3. Golgi 4. Plasma membrane

8. Genetics: Schekman and co-workers Novick et al. 1980: Identification of 23 SEC genes

9. Genetics: Schekman and co-workers

10. In vitro assay for vesicular transport: Rothman and co- workers Balch et al. 1984

11. In vitro assay for ER translocation: Blobel and co-workers

13. Identification of signal sequences

14. SRP: signal recognition particle

16. SRP cycle

18. Site-specific incorporation of modified amino acids to probe ER translocation

19. ER translocation can be reconstituted in vitro: - Sec61 complex: conserved translocation channel Sec61 subunits (a, b, g) Sec62/63 TRAM (translocating chain-assoc. membrane protein) - phospholipids (proteoliposomes) and luminal chaperones (BIP) - SRP/SRP receptor only required for co-translational translocation not for post-translational translocation (e.g pre-pro-alpha factor). - energetics of translocation: protein conducting channel (cotranslational) molecular ratcheting (posttranslational)

20. Probing of translocation intermediates with fluorescent peptides From: Liao and Johnson Cell (97)

21. The Sec61 complex forms a channel…. Menetret et al. Mol Cell (2000) 6:1219

22. From: Beckmann et al. Cell (2001) Vol 107, 361-372 The translocon by EM tomography

23. From: Beckmann et al. Cell (2001) Vol 107, 361-372 The translocon by EM tomography

24. From: Van den Berg et al. Nature (2004) 427, 36-44 X-ray structure of the prokaryotic translocation channel SecY

25. From: Van den Berg et al. Nature (2004) 427, 36-44 Model for translocation of soluble proteins

26. Topology of membrane-spanning proteins

27. Type I membrane proteins have a cleavable signal sequence

28. Type II membrane proteins have internal signal sequence

29. Type III membrane proteins have internal signal sequence

30. Type II+III membrane proteins have internal signal sequences

31. From: Beckmann et al. Cell (2001) Vol 107, 361-372 Model for translocation of transmembrane proteins

32. Translocation of proteins with multiple membrane spanning domains