The Secretory Pathway Becky Dutch Molecular and Cellular Biochemistry 1. ER - translation 2. ER- protein modifications

1. The Secretory Pathway Becky Dutch Molecular and Cellular Biochemistry 1. ER - translation 2. ER- protein modifications 3. Discussion Section 4. Golgi apparatus 5. Vesicular transport

2. Lecture 5: Transport Vesicles Reading: Alberts Chapter 13 Lodish Sections 17.10

3. Vesicular Transport 1. How are these vesicles formed? How are different proteins incorporated? 2. How are these vesicles targeted? 3. How do they fuse with their target? . Lodish, Fig 17-13

4. Three types of coated vesicles known: Each have different coat protein: clathrin COP I COP II Each involved in specific cellular transport pathways Lodish 17-50

5. Types of vesicles and their target locations Lodish 17-51 All three types have similar vesicle budding: Coat proteins polymerize around the cytosolic face of budding vesicle Coat and adapter proteins help select cargo GTP-binding protein regulates the rate of vesicle formation

6. COP I vesicles Coat protein formed from coatamers: cytosolic complex with seven subunits. Polymerize on surface of vesicles to drive formation. Dissociate from vesicle after formation. Golgi transport - retrograde and likely anterograde Also retrograde Golgi to ER transport Lodish 17-56

7. Cell-free system for studying Golgi transport Cultured cells missing one of the processing enzymes - this will allow differentiation of the two populations of Golgi Lodish 17-57

8. Cell-free system for studying Golgi transport Infect mutant cells with VSV - makes only one viral glycoprotein - VSV G Addition of N-acetylglucos. to VSV G wlll only happen if transport to wt Golgi stack occurs Assay used to identify and study function of proteins involved in Golgi vesicular transport Lodish 17-57

9. Formation of COP I vesicles Cell-free system just described very helpful in determing roles 1. ARF - small GTPase, releases GDP and binds GTP - Golgi attached enyzme that promotes this unknown 2. ARF-GTP binds receptors on Golgi membrane 3. COP I coatamers bind to ARF, other protein on cytosolic face. 4. Fatty acyl CoA helps budding mechanism unknown. 5. If non-hydrolyzable GTP used - vesicles form and release, but COP I never disassociates Lodish 17-58

10. Role of COP I vesicles Retrograde transport - Golgi to ER KDEL receptor and other membrane proteins to be returned to ER - have KKXX sequence at end of C-terminus. This binds COP a and b. This sequence necessary and sufficient to drive transport to ER. Yeast mutants lacking COP a and b can’t do retrograde transport Retrograde transport - in Golgi Moving specific proteins trans to medial, medial to cis Anterograde transport in Golgi COP I vesicles with lots of cargo, no KDEL - fast track

11. COP II vesicles ER to Golgi transport Cell-free extracts of yeast rough ER plus cytosol and ATP - vesicles form - COP II Formation - similar to COP I. Sec12 catalyzes exchange of GDP for GTP in th Sar I protein. Complex forms with Sec23 and Sec24 proteins, followed by binding of Sec13, Sec31, then Sec16. Contain a family of 24kDa proteins that selectively bind soluble proteins bound for Golgi. Integral membrane proteins to be transported generally have Asp-X-Glu sequence - binds to one or more COP II proteins

12. Exocytosis: TGN to Cell Surface Constituitive and Regulated secretion Clathrin vesicles Alberts 13-36

13. Exocytosis of secretory vesicles Secretory vesicles very densely packed - can release large amounts of material Regulated secretion - vesicles move from TGN to site of secretion. Can be a long distance (nerve cells) Triggered release - signal to secrete can be hormone binding receptor, electrical excitation. Increases in Ca2+ often important. Alberts 13-39

14. Mast cell - example of regulated exocytosis Histamine released in response to binding of specific ligands Gives many of symptoms of allergic reactions Mast cell incubated in solution with ligand -Response all over cell If ligand is localized to one spot - response will be localized. Alberts 13-41

15. Targetting and Fusion Common motifs for all types of vesicles - fusion after depolymerization; conserved set of proteins that promote targetting and fusion V-SNARE - in transport vesicle - important for targeting T-SNARE - on target, along with ubiquitous SNAP-25 V-SNARE, T-SNARE, SNAP25 form complex - fusion Other proteins involved - NSF (ATPase), SNAP proteins Lodish 17-59

16. Rab proteins - regulators of vesicular traffic Rab proteins - GTP binding proteins Approx. 200 amino acids - structure similar to Ras Bind and hydrolyze GTP - this is hypothesized to regulate rate of vesicular fusion GDI - catalyzes GDP/GTP exchange of Rabs - this leads to conformational change in Rab that lets it bind vesicle GTP hydrolysis leads to release of Rab after membrane fusion

17. Structures of many of these proteins recently determined Brunger, Curr. Op. Struct. Biol. (2001) 11:163-173 Synaptobrevin=VAMP = v-SNARE; syntaxin=t-SNARE; synaptotagmin - Ca2+ binding protein; SNARE complex = portions of synaptobrevin, syntaxin and SNAP-25

18. SNARE complex has several states - zipper model A - closed state; B - binary - syntaxin, SNAP25; C; D - ternary - with synaptobrevin in complex

19. Viral fusion proteins - best-understood examples Single protein systems which promote high level membrane fusion Syncytia assay of wt SV5 F and the F Tail- mutant.

20. Viral fusion proteins undergo conformational changes upon triggering of fusion Lodish 17-60

21. Similar complexes containing heptad repeat regions form in a number of viral fusion proteins

22. Formation of these heptad repeat complexes critical for membrane fusion

23. Steps for fusion pore formation A group of pH activated HA spikes work together to form fused membranes HA protein inactive after this process - unlike SNARES, which recycle Lodish 17-61

24. Relation of SNARE to viral fusion proteins Complexes containing coiled-coils fundamental to both systems Skehal and Wiley, Cell (1998) 95: 871-874

25. Secretory pathway - critical for cellular function . Lodish, Fig 17-13