1. Vesicular Trafficking Movement From the ER Through the Golgi

2. Getting to the Golgi Anterograde and Retrograde Traffic

3. Genetic Analysis of ER-Golgi Transport

4. General Principles Cargo selection –signals (ie amino acid sequences) Vesicle formation (budding) from donor compartment Vesicle/Cargo targeting and fusion to acceptor compartment Most regulation is mediated by cargo- and target-compartment-specific small GTPases

5. Anterograde transport –COPII apparatus –protein complex mediating cargo selection and assembly of budding components from ER Retrograde transport –COPI –protein complex mediating transfer of vesicles back to ER

6. Retrograde Transport Gives Golgi Compartments Dynamic Identity

7. Cargo Selection: Export Targeting Sequences Y x n DxE – tyrosine-diacidic domain Found in cytoplasmic tail of TM proteins

8. Cargo Selection: Just Getting the Right Stuff Exclusion of ER resident proteins –not well understood –fidelity factors (genes) mutations in some genes cause increased excretion of ER resident proteins –Retrieval COPI vesicles retrieve ER proteins and return them KDEL signal on lumenal proteins KKxx-COOH on TM proteins (dilysine motif)

9. COPII Vesicular Coat Assembly & Budding COPII coat formation regulated by sar1 GTPase sar1 activates cargo receptors –TM proteins recognizing export signals sar1 recruits sec23/24 coatamer proteins sec23/24 in turn recruit sec31/13 coat proteins COPII vesicles form and bud Once apart from ER, coat proteins are triggered to release by hydrolysis of GTP by sar1

10. Vesicular Delivery Rab-GTPases Recruits tether and fusion proteins into vesicles These form a targeting complex recognized by a docking complex targeting –v-tethers – p115 –vSNAREs – synaptobrevin docking –t-tethers – GRASP65 –tSNAREs – syntaxin 1

11. Tethering and Fusion Mediated by SNAREs (SNAP Receptors) SNAREs aggregated by Rab-GTPase (vSNAREs) Vesicle buds SNAP25-NSF interact with vesicle to dis-aggregate and prime vSNAREs for interaction with tSNAREs

12. COPI Vesicles Recycle ER Resident Proteins COPI Vesicles –Arf family of small GTPases –Recruit/activate coatomer (6 cytosolic proteins) –Recruit ER targeting SNAREs

13. Retrieval of ER Proteins with KDEL Signal by COPI Vesicles

14. Cycle of Vesicular Transport

15. Cargo Sorting by the Trans-Golgi Network (TGN) Specific coat proteins direct TGN vesicles to targets

16. Lysosomal/Endosomal Targeting Mannose-6-phosphate receptor targeting to lysosome/endosome MPR cytoplasmic tail (C-term) has multiple signals for multiple routing possibilities

17. Lysosomal Targeting of Prohydrolase by M6P and MPRs Mannose-6-Phosphate Receptors Cation-independent (CI) Cation dependent (CD)

18. Cytoplasmic Tails of MPRs Allow Sorting to Lysosomes vs Other Routes

19. Assembly of Clathrin on Lysosomal/Endosomal Destined TGN Vesicles

20. Directed Translocation of Secretory Vesicles apical targeting in caveolin coated, lipid- raft vesicles basolateral targeting signals –direct protein to this membrane constitutive basolateral targeting –subsequent sorting of apical proteins untargeted vesicles –sorting to redistribute surface proteins

21. Proteolytic Processing of Pro-proteins in Secretory Vesicles Cleavage of proinsulin in secretory vesicles of pancreas cells

22. Secretory Vesicles/Granules Material in secretory vesicle aggregates due to decreased pH and divalent cations Membrane recycling to TGN directly or via endosome reduced vesicle size

23. Regulated Secretion Fusion of the vesicle and plasma membrane is regulated by various means –receptor-ligand interaction and second messenger signaling –depolarization of membrane –etc…