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Vesicular Trafficking

Vesicular Trafficking. Movement From the ER Through the Golgi. Getting to the Golgi. Anterograde and Retrograde Traffic. Genetic Analysis of ER-Golgi Transport. General Principles. Cargo selection signals (ie amino acid sequences) Vesicle formation (budding) from donor compartment

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Vesicular Trafficking

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

  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…

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