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

• Vesicle/Cargo targeting and fusion to acceptor compartment

• Most regulation is mediated by cargo- and target-compartment-specific small GTPases

• 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

Retrograde Transport Gives Golgi Compartments Dynamic Identity

Cargo Selection: Export Targeting Sequences

• Y xnDxE – tyrosine-diacidic domain• Found in cytoplasmic tail of TM proteins

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)

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

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

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

COPI Vesicles

Recycle ER Resident Proteins

• COPI Vesicles– Arf family of small GTPases– Recruit/activate coatomer (6 cytosolic proteins)– Recruit ER targeting SNAREs

Retrieval of ER Proteins with

KDEL Signal by COPI Vesicles

Cycle of Vesicular Transport

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

Lysosomal/Endosomal Targeting

• Mannose-6-phosphate receptor targeting to lysosome/endosome

• MPR cytoplasmic tail (C-term) has multiple signals for multiple routing possibilities

Lysosomal Targeting of

Prohydrolase by M6P and MPRs

•Mannose-6-Phosphate Receptors •Cation-independent (CI)•Cation dependent (CD)

Cytoplasmic Tails of MPRs Allow Sorting to Lysosomes vs Other Routes

Assembly of Clathrin on Lysosomal/Endosomal Destined TGN Vesicles

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

Proteolytic Processing of Pro-proteins in Secretory

Vesicles

• Cleavage of proinsulin in secretory vesicles of pancreas cells

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

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…