INTD5000, Lectures C7 & C8 Professor Eileen M. Lafer Tel#: 7-3764 Email: Lafer@biochem.uthscsa

1. INTD5000, Lectures C7 & C8 Professor Eileen M. Lafer Tel#: 7-3764 Email: Lafer@biochem.uthscsa.edu Office: Room 415B THE SECRETORY AND ENDOCYTIC PATHWAYS Reading: Chapters 12 and 13 from Alberts et al., Molecular Biology of the Cell

2. CELL COMPARTMENTS (ANIMATION 12.1 ALBERTS)

3. THREE TYPES OF PROTEIN MOVEMENT BETWEEN COMPARTMENTS Gated transport: nuclear pore complex, cytosol<-> nucleus Transmembrane transport: protein translocators (proteins usually unfold), cytosol-> ER, cytosol-> mitochondria Vesicular transport: membrane-enclosed transport, ER <-> golgi post-golgi traffic Alberts

4. THE BIOSYNTHETIC-SECRETORY AND ENDOCYTIC PATHWAYS Alberts

5. ENDOPLASMIC RETICULUM (ER): An extensive tubovesicular network where proteins and lipids are made. Rough ER: studded with ribosomes, site of protein biosynthesis Smooth ER: site of lipid biosynthesis Alberts

6. ROUGH ER Alberts

7. SMOOTH ER Alberts

8. Alberts

9. PROTEIN SYNTHESIS AND TRANSLOCATION IN THE ROUGH ER Animation 12.6, Alberts

10. PROTEIN GLYCOSYLATION IN THE ROUGH ER: During translation, a signal sequence on membrane and secretory proteins directs the nascent protein into the ER lumen. After the protein has entered the ER, the glycosylation process begins.

11. A PRE-FORMED PRECURSOR OLIGOSACCHARIDE IS TRANSFERRED EN BLOC TO PROTEINS IN THE ER Alberts

12. PROTEIN GLYCOSYLATION IN THE ROUGH ER Alberts

13. SOME PERIPHERAL MEMBRANE PROTEINS AQUIRE A COVALENTLY ATTACHED GLYCOPHOSPHATIDYLINOSITOL (GPI) ANCHOR IN THE ER Alberts

14. THE BIO-SYNTHETIC SECRETORY AND ENDOCYTIC PATHWAYS Retrieval pathways are in blue. Alberts

15. MOVIE: MOVEMENT OF A FLUORESCENTLY TAGGED MEMBRANE PROTEIN THROUGH THE SECRETORY PATHWAY Alberts Video 13.2

16. EXOCYTOSIS AND ENDOCTYOSIS Alberts

17. VESICULAR TRANSPORT Transport vesicles bud from one compartment and fuse with another, carrying material from the lumen of the donor compartment, and depositing it in the lumen of the target compartment. Alberts

18. VESICULAR TRANSPORT IS MEDIATED BY COATED VESICLES Alberts et al., Molecular Biology of the Cell-3rd edition

19. THERE ARE VARIOUS TYPES OF COATED VESICLES Alberts

20. DIFFERENT COATS ARE USED IN DIFFERENT TRAFFICKING PATHWAYS Alberts

21. CLATHRIN-COATED VESICLES ARE THE MOST WELL CHARACTERIZED TRANSPORT VESICLES John Heuser

22. THE STRUCTURE OF A CLATHRIN TRISKELION Ungewickell and Branton

23. THE STRUCTURE OF A CLATHRIN COAT Kirchhausen, Harrison, Walz, Fotin

24. The Assembly and Disassembly of a Clathrin Coat

25. AP-2 a b AP180 m C N s Clathrin assembly Clathrin assembly THE CLATHRIN ASSEMBLY PROTEINS Monomeric AP Family Tetrameric AP Family (also called adaptins) AP180 Synaptic Plasma Membrane AP-1 TGN CALM Ubiquitous AP-2 Plasma Membrane AP-3 Endosome/Lysosome AP-4 TGN PIP binding PIP binding

26. Adaptins (AP-1, AP-2, AP-3, AP-4) SelectCargo For Inclusion Into Coated Vesicles, and Promote Clathrin Polymerization YXXF-m2 LL-b1 NPXY-clathrin TD Alberts et al., Molecular Biology of the Cell

27. AP180 Promotes the Formation of Homogeneously Sized Vesicles Clathrin Cages Assembled in vitroWithoutAP180 Synaptic Vesicles in Drosophila Lacking Fly AP180 Gene lap Clathrin Cages Assembled In vitro WithAP180 Synaptic Vesicles in Wild Type Drosophila Containing Fly AP180 Gene lap Ye & Lafer, 1995 Zhang, Koh, Beckstead, Budnick, Ganetzky, & Bellen, 1998

28. AP180 recruits clathrin to the membrane via interactions with the phosphoinositide PIP2, and stimulates coat formation. Diameter of lattice: 66nm Matthew Higgins and Harvey McMahon

29. THE GTPase DYNAMIN PROMOTES MEMBRANE SCISSION Alberts

30. How do clathrin coated vesicles uncoat? Hsc70 auxilin clathrin APs Ca++

31. How do clathrin coated vesicles uncoat? Disrupt Interaction Between Hsc70 and Auxilin Hsc70 Auxilin-DHPD clathrin APs Ca++

32. Auxilin DHPD Increases the Number of Coated Vesicles by 6 Fold: Auxilin Auxilin DHPD * * * * J.R. Morgan, K. Prasad, S. Jin, G.J. Augustine, and E.M. Lafer. Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc70 and Auxilin. Neuron32: 289-300 (2001).

33. Auxilin Recruits and Activates the Uncoating ATPase Hsc70 to Clathrin Coated Vesicles Hsc70 auxilin clathrin APs Ca++

34. The Clathrin Coated Vesicle Cycle

35. CLATHRIN – THE MOVIE ANIMATION 13.1 ALBERTS

36. ASSEMBLY AND BUDDING OF COPII and COPI COATED VESICLES FROM: Lee et al., Annu. Rev. Cell Dev. Biol. 20:87-123, 2004.

37. STRUCTURE OF THE SEC13/31 COPII COAT FROM: Stegg et al., Nature 439: 234-239, 2006.

38. MOVIE: STRUCTURE OF THE SEC13/31 COPII COAT FROM: Stegg et al., Nature 439: 234-239, 2006.

39. ORIENTATION OF THE SEC13/32 HETEROTETRAMER IN THE SELF-ASSEMBLED OCTAHEDRAL CAGE FROM: Stegg et al., Nature 439: 234-239, 2006.

40. SNARE PROTEINS CONTRIBUTE TO THE SELECTIVITY OF VESICLE-TARGET DOCKING AS WELL AS TO THE FUSION PROCESS

41. SNARE PROTEINS: • The group of proteins commonly referred to as SNARES for • Soluble NSF Attachment REceptorS • were originally discovered as the synaptic proteins: • VAMP/synaptobrevin • syntaxin • SNAP-25 • They were later re-discovered as the receptors for the soluble • Golgi trafficking protein SNAP – Soluble NSF Attachment Protein. • NSF (NEM-Sensitive Fusion factor) • The SNAREproteins were also identified as substrates for the clostridial neurotoxins, potent agents which inhibit neurotransmitter release.

42. The SNARE Complex: • The synaptic vesicle membrane protein synaptobrevin (v-snare), forms a tight ternary complex with the presynaptic plasma membrane proteins syntaxin (t-snare), and SNAP-25 (t-snare). The stoichiometry of the proteins in the complex is 1:1:1 and it is resistant to SDS. This complex can be actively disassembled by the ATPase NSF, together with -SNAP.

43. FROM: Sutton et al., Nature 395: 347-353, 1998. Topology and organization of the synaptic fusion complex. a, Backbone ribbon drawing of the synaptic fusion complex: blue, synaptobrevin-II; red, syntaxin-1A; green, SNAP-25B (Sn1 and Sn2).

44. Hypothetical model of the synaptic fusion complex as it joins two membranes, and location of neurotoxin-mediated cleavage sites. We extended the synaptic fusion complex crystal structure to include the transmembrane domains (yellow) of syntaxin-1A (red) and synaptobrevin-II (blue), and the loop connecting the Sn1 and Sn2 fragments (green). The transmembrane domains and the linker to the Sx fragment are represented as a-helices. Hypothetical bends of the syntaxin and synaptobrevin a-helices were modelled close to the lipid bilayers. The loop between the Sn1 and Sn2 fragments was modelled as an unstructured polypeptide chain. The conformation of this loop is speculative. The loop between the Sn1 and Sn2 domains is shown in orange.

45. A MODEL FOR HOW SNARE PROTEINS MAY CATLYZE MEMBRANE FUSION Alberts

46. DISSOCIATION OF SNARE PAIRS BY NSF FOLLOWING A CYCLE OF MEMBRANE FUSION Alberts

47. RAB PROTEINS ARE SMALL GTPases THAT GUIDE VESICLE TARGETING Alberts

49. TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS Alberts

50. PROTEINS LEAVE THE ER IN COPII-COATED TRANSPORT VESICLES