Vesicular Trafficking Flashcards
Secretory Pathway
ER –> Golgi –> Organelle/Plasma membrane
Trans Golgi Network sorting
Constitutive vs Regulated
Constitutive:
- Secretory proteins are continuously secreted (serum proteins)
- Transported in vesicles directed to plasma membrane for exocytosis
Regulated:
- Storing proteins in secretory vesicles awaiting a stimulus for exocytosis
- e.g. Insulin secretion in Pancreatic B cells (also neurotransmitters)
- > Calcium influx triggers fusion of vesicles with membrane and exocytosis
Studying the secretory pathway: VSV-G Trafficking
Vesicular Stomatitis Virus G-protein = abundant membrane glycoprotein with a temperature sensitive mutant that can be labelled
- Can Switch ON/OFF transport using temperature and track the pathway from labelling
Studying the secretory pathway: Endo-D Cleavage
Modifying membrane with mannose trimming to become sensitive to Endo-D enzyme cleavage
- ER resident proteins = untrimmed (full oligosaccharide complement present) and therefore insensitive to Endo-D
- Cleaved (Golgi) and Uncleaved (ER) forms distinguished by size on SDS-PAGE
- Proteins must be radiolabelled at 40 degrees to prevent correct folding (return to 32 degrees for correct folding)
Assembly of vesicle protein coat
Drives vesicle formation and cargo molecule selection
- Driven by polymerisation of soluble proteins complexes on the cytosolic side of the protein
- Provides curvature and acts as a filter regulating other proteins that can be added
- Can disassemble to leave a completed transport vesicle
Vesicle formation depends on:
- Shapes of phospholipids when 2 bilayer faces are asymmetric
- Large curved proteins (scaffold and bend membrane)
- Insertion of amphipathic a-helices into one side of bilayer
- Oligomerization of several monomers scaffolding and curving membrane
- High surface concentration of membrane-binding proteins: adds steric pressure to bend bilayer
Small GTP-binding proteins regulating vesicle coat assembly (COPI & COPII)
COPI & Clathrin = ARF1
COPII = SAR1
SAR1 GTP-binding protein
Needed for COPII vesicle assembly
1 - Sec12 catalyses GTP binding to SAR1: causes conformation change to expose hydrophobic N-terminus
2 - Membrane-attached SAR1 drives polymerisation of COPII vesicles acting as a binding site for the Sec23/24 complex
3 - Sec23 promotes GTP hydrolysis of SAR1
4 - Release of SAR1-GDP causes coat disassembly
Vesicle coats selecting cargo proteins
Targeting sequences on cargo proteins are selected for by vesicle coat
- COPII coat: ternary complex between SAR1, Sec23 and Sec24
- Target sequence = Diacidic motifs in COPII cargo
Guiding vesicles to target membranes:
SNARE proteins
v-SNAREs (vesicular)
- e.g. Synaptobrevin
- Incorporated into the membranes of transport vesicles during budding
- a.k.a. R-SNAREs: contribute an Arginine (R) residue
t-SNAREs (target)
- e.g. Synbaxin or SNAP-25
- Associated with target membranes
- a.k.a. Q-SNAREs: contribute a Glutamine (Q) residue
Guiding vesicles to target membranes:
GTPases, Rab proteins
GTPases play important role in regulating vesicle formation, movement along cytoskeleton and fusion to target.
Activated Rab enters membranes and recruits effector proteins:
1 - Rab-GDP present in cytosol bound to GDP Dissociation Inhibitor (GDI) preventing activation
2 - At appropriate location, GDI-Displacement Factor (GDF) displaces GDI
3 - With GDI displaced, Guanine Exchange Factor (GEF) can activate Rab by exposing its prenyl group (hydrophobic) to bind to membrane
4 - Rab-GTP can bind Rab effector proteins to recruit specific proteins
5 - GTP hydrolysis releases Rab to bind to GDI again
Early Stages of Secretory Pathway
Anterograde
- COPII vesicles
- ER –> Golgi
Retrograde
- COPI vesicles
- Golgi –> ER
Later stages of Secretory Pathway
Cis Golgi –> Trans Golgi (Oligosaccharide modifications)
- COPI retrograde transport from Trans to Cis
- Glycosyl transferases maintained at sufficient levels in Cis-Golgi (O-linked glycosylation)
- TGN for protein sorting
O-linked glycosylation
Adding a large glycan (sugar) to an exposed Oxygen atom of protein in phosphorylated (active) form
- Occurs exclusively in Cis Golgi
- Mediated by Glycosyl Transferases
Clathrin structure
A triskelia composed of 3 clathrin heavy chains and 3 light chains:
- Triskelia interact with each other to form a polyhedral lattice that surrounds the vesicle
Clathrin Coated Vesicles (CCV) distribution
Selectively sorts cargo at the:
- cell membrane
- TGN
- endosomal compartments
Clathrin-associated adaptors
Adaptor Proteins AP-1, AP-2 and AP-3 recognise cargo protein’s sorting signals. GGA adaptor proteins also have clathrin/cargo binding elements.
- Signals are usually tyrosine (bulky hydrophobic residue included) or dileucine (downstream of acidic residue) based
- All vesicles containing AP coats use GTPase ARF to initiate coat assembly from donor membrane (ARF is also used in COPI retrograde transport)
Dynamin helps pinching off of vesicles
Upon bud formation, dynamin polymerises around vesicle neck
- Using energy from GTP hydrolysis, dynamin undergoes conformational change until piching off
- observed in nerve terminals
Vesicle uncoating
Clathrin coated pits uncoat allowing the triskelion to be re-used
Uncoating allows exposure of v-SNAREs needed for vesicle docking
Golgi to Lysosome transport
Driven by Mannose-6-Phosphate (M-6-P) phosphorylating lysosome-specific proteins (functional proteins!) on their specific mannose sugars:
1) M6PR binds M6P at slightly acidic pH 6.5 in Golgi
2) M6P recognised by M6PR in clathrin/AP1 vesicles which bud off
3) After budding, coat rapidly depolymerises and fuses with late endosome
4) Endosome’s lower pH (5-5.5) causes M6PR to release M6P thus releasing cargo.
Receptor mediated endocytosis
Triggered by ligand binding to receptor
- promotes movement of receptor-ligand complex to clathrin-coated pits where endocytosis can occur
Transferrin Receptor (TfR)
Transferrin receptor expressed on cell surface, required for importing iron (regulated by intracellular iron concentrations)
- imports transferrin-iron complex by endocytosis
LDL receptor
Cell surface receptor recognises and binds cholesterol in blood stream before being endocytosed
- In endosome, LDL dissociates from receptor for hydrolytic breakdown for cellular use
- LDL reeceptor recycled back to cell surface
Epidermal Growth Factor (EGF) receptor
Unlike LDL receptors, ligand binding promotes receptor dimerization, endocytosis and lysosomal degradation (NO recycling)
