1. Lecture 3 Vesicular Trafficking -Cops and Clathrins -Arfs, Rabs, Sars -Snares

2. Vesicular Trafficking allows Proteins and Vesicles to Reach their Destinations

3. The orientation of transmembrane proteins Proteins IN the ER face the OUTSIDE of the cell

4. Exocytosis Vescles are transported along the Actin Cytoskeleton

5. COPII = ER-> Golgi COPI = Golgi to Golgi

6. Vesicular Trafficking allows Proteins and Vesicles to Reach their Destinations

7. The orientation of transmembrane proteins Proteins IN the ER face the OUTSIDE of the cell

8. COPII = ER-> Golgi COPI = Golgi to Golgi

10. Vesicle budding and vesicle fusion are two different processes Both mechanically and mechanistically

11. Triskeleion = 3 large and 3 small polypeptides = 1 clathrin Receptor-mediated Endocytosis requires clathrin

13. Adaptin binds clathrin and receptors, acting as a bridge (4 types) Hsp70 chaperone and auxillin uncoat the vesicle. What prevents uncoating at membranes? Vesicles form at many membranes (Golgi, PM) At these membranes COPI and COPII function instead of clathrin Vesicles can be tubular

14. Dynamin forms a ring around the bud (GTPase)

15. GAP = GTPase Activating Protein GEF = Guanine Nucleotide Exchange Factor GTPases function in vesicle formation

16. GTPases are required for vesicle formation (start) and Vesicle fusion (end) ARF is the GTPase for COPI and Sar is the GTPase for COPII GEFs determine when the vesicle is ready to bud GEF activation triggers GTPase activation and hydrophobic tail exposure GAPs triggers GTPase inactivation. It falls off the membrane and triggers coat disassembly

17. Snares guide vesicular transport Vesicle-surface markers that direct vesicles to the correct place V=vesicle and t=target SNARES

18. Snares are integral-membrane proteins that pull membranes together. Neuronal snares are the targets of neural toxin proteases (botulism) The coiled coil The coiled-coil is a tightly intertwined set of 4  -helix domains Three are contributed by t-snares, and 1 by the v-snare At least 1 of the t-snares is an integral-membrane protein Snares (20) and Rabs

19. Snares also promote Membrane fusion

20. 30 members -each bind a particular vesicle -On the cytoplasmic face Rabs interact with Snares Variation in Rab C-terminal tails Variation in effectors Rabs are different from coat Assembly GTPases (ARFs) Rab GTPases ensure the specificity of vesicular docking

21. We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking. SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1– positive Early Endosome/Trans-Golgi Network Waka Natsume et al.

22. How are Snares separated? NSF is an ATPase that dissociates Snare pairs

23. HIV enters through membrane fusion Influenza enters through receptor-mediated endocytosis Snare-like IM-like Proteins Hydrophobic Tails exposed