Vesicular Trafficking Flashcards Preview

Biological Membranes and Signalling > Vesicular Trafficking > Flashcards

Flashcards in Vesicular Trafficking Deck (24)
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1
Q

Secretory Pathway

A

ER –> Golgi –> Organelle/Plasma membrane

2
Q

Trans Golgi Network sorting

Constitutive vs Regulated

A

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
3
Q

Studying the secretory pathway: VSV-G Trafficking

A

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
4
Q

Studying the secretory pathway: Endo-D Cleavage

A

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)
5
Q

Assembly of vesicle protein coat

A

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
6
Q

Vesicle formation depends on:

A
  • 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
7
Q

Small GTP-binding proteins regulating vesicle coat assembly (COPI & COPII)

A

COPI & Clathrin = ARF1

COPII = SAR1

8
Q

SAR1 GTP-binding protein

A

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

9
Q

Vesicle coats selecting cargo proteins

A

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
10
Q

Guiding vesicles to target membranes:

SNARE proteins

A

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
11
Q

Guiding vesicles to target membranes:

GTPases, Rab proteins

A

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

12
Q

Early Stages of Secretory Pathway

A

Anterograde

  • COPII vesicles
  • ER –> Golgi

Retrograde

  • COPI vesicles
  • Golgi –> ER
13
Q

Later stages of Secretory Pathway

A

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
14
Q

O-linked glycosylation

A

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
15
Q

Clathrin structure

A

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

16
Q

Clathrin Coated Vesicles (CCV) distribution

A

Selectively sorts cargo at the:

  • cell membrane
  • TGN
  • endosomal compartments
17
Q

Clathrin-associated adaptors

A

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)
18
Q

Dynamin helps pinching off of vesicles

A

Upon bud formation, dynamin polymerises around vesicle neck

  • Using energy from GTP hydrolysis, dynamin undergoes conformational change until piching off
  • observed in nerve terminals
19
Q

Vesicle uncoating

A

Clathrin coated pits uncoat allowing the triskelion to be re-used

Uncoating allows exposure of v-SNAREs needed for vesicle docking

20
Q

Golgi to Lysosome transport

A

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.

21
Q

Receptor mediated endocytosis

A

Triggered by ligand binding to receptor

  • promotes movement of receptor-ligand complex to clathrin-coated pits where endocytosis can occur
22
Q

Transferrin Receptor (TfR)

A

Transferrin receptor expressed on cell surface, required for importing iron (regulated by intracellular iron concentrations)
- imports transferrin-iron complex by endocytosis

23
Q

LDL receptor

A

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
24
Q

Epidermal Growth Factor (EGF) receptor

A

Unlike LDL receptors, ligand binding promotes receptor dimerization, endocytosis and lysosomal degradation (NO recycling)