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

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 Alberts Retrieval pathways are in blue.

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 Alberts 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.

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. Ungewickell and Branton THE STRUCTURE OF A CLATHRIN TRISKELION

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

24. The Assembly and Disassembly of a Clathrin Coat

25. Monomeric AP Family Tetrameric AP Family (also called adaptins) Clathrin assembly N C AP180AP-2     Clathrin assembly AP180Synaptic Plasma MembraneAP-1TGN CALMUbiquitousAP-2Plasma Membrane AP-3Endosome/Lysosome AP-4TGN PIP binding THE CLATHRIN ASSEMBLY PROTEINS

26. Adaptins (AP-1, AP-2, AP-3, AP-4) Select Cargo For Inclusion Into Coated Vesicles, and Promote Clathrin Polymerization Alberts et al., Molecular Biology of the Cell YXX  LL  NPXY-clathrin TD

27. AP180 Promotes the Formation of Homogeneously Sized Vesicles Clathrin Cages Assembled in vitro Without AP180 Clathrin Cages Assembled In vitro With AP180 Synaptic Vesicles in Drosophila Lacking Fly AP180 Gene lap 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. Matthew Higgins and Harvey McMahon Diameter of lattice: 66nm

29. THE GTPase DYNAMIN PROMOTES MEMBRANE SCISSION Alberts

32. Auxilin  HPD Increases the Number of Coated Vesicles by 6 Fold: Auxilin * * * * Auxilin  HPD 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. Neuron 32: 289-300 (2001).

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 SNARE proteins 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  -helices. Hypothetical bends of the syntaxin and synaptobrevin  -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

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

51. ER RESIDENT PROTEINS CONTAIN A KDEL SEQUENCE THAT INTERACTS WITH THE COPI COAT AND PERMITS THEIR RETRIVAL FROM THE GOLGI BACK TO THE ER

52. A MODEL FOR THE RETRIEVAL OF SOLUBLE ER RESIDENT PROTEINS

53. THE GOLGI APPARATUS CONSISTS OF AN ORDERED SERIES OF COMPARTMENTS Alberts

54. THE TRANSITIONAL ZONE BETWEEN THE ER AND GOLGI Alberts

55. THE GOLGI APPARATUS Alberts

56. OLIGOSACCHARIDE CHAINS ARE PROCESSED IN THE GOLGI APPARATUS

57. TRANSPORT INTO THE CELL FROM THE PLASMA MEMBRANE: ENDOCYTOSIS

58. PINOCYTIC VESICLES FORM FROM COATED PITS IN THE PLASMA MEMBRANE 1. The major type of endocytic vesicles are the clathrin coated vesicles (CCVs). The CCVs shown below are from a hen oocyte taking up lipoprotein particles to form yolk: Alberts

59. 2.Caveolae are a less well understood type of endocytic coated vesicle that are involved in the endocytosis of lipid rafts from the plamsa membrane. Their major structural proteins are caveolins which are integral membrane proteins. Alberts Alberts

60. ENDOCYTOSIS IS IMPORTANT FOR CELLS TO: 1.Import selected extracelluar molecules (i.e. receptor-mediated endocytosis). 2.Regulate levels of membrane proteins on the cell surface (i.e. receptor down-regulation). 3. Synaptic vesicle recycling and biogenesis.

61. EXAMPLE: RECEPTOR MEDIATED ENDOCYTOSIS Cholesterol molecules are packaged in LDL particles. Alberts

63. RECEPTOR-MEDIATED ENDOCYTOSIS: THE MOVIE Animation 13.3 from Alberts

64. EXOCYTOSIS Alberts

65. SECRETORY VESICLES BUD FROM THE TRANS-GOLGI Alberts

66. EXOCYTOSIS OF SECRETORY VESICLES The release of insulin from a secretory vesicle of a pancreatic beta cell. Alberts

67. SYNAPTIC TRANSMISSION IS AN EXAMPLE OF REGULATED EXOCYTOSIS SYNAPTIC TRANSMISSION IS ALSO AN EXAMPLE OF A COUPLED EXO- ENDOCYTIC CYCLE Augustine