Endomembrane system ALL

Question 1

What is membrane trafficking

Answer 1

• Process where proteins + other macromolecules are distributed throughout the cell
• Uses membrane-bound vesicles as transport int.
• One pathway = secretory pathway to ER for exocytosis
• Another = endocytic pathway

Question 2

Methods for studying trafficking

Answer 2

1. Imaging (light microscopy is structures are poor, e- microscopy for unknown structures)
2. Pulse-chase imaging (e.g. radioactive methionine, cytosol → ER, silver nitrate precipitates, add unlabelled, RUSH)
3. Genetics (SEC screen, vacuole protein screen, autophagy screen)
4. Biochemistry (in vitro reconstitution, follow transport, 2 populations of Golgi fused together, donor membrane isolated from mutant, vast majority or glycoprotein x incorporate GlcNac as moves through Golgi acceptor derived from WT, GlcNac incorporated into viral protein from donor to acceptor compartment

Question 3

Journey through the secretory system

Answer 3

1. PTM (disulphides only formed in ER, evolution, cells secreted, Ero1)
2. N-linked glycosylation (oligosacch. transferase catalyses 1st stage in glycosylation, specificity of glycosylating E are affected by the residues surrounding the patterns

Question 4

Quality control in the ER

Answer 4

Glucosidase cycle

• Calnexin binds incompletely folded protein, traps in ER
• Glucosidase removes terminal glucose
• Glycosyl transferase determines if protein folded
• If many unusual folds, mannosidase removes terminal mannose → signal for protein to be exported from ER to cytoplasm for degradation
• ER associated degradation (calnexin + lectin, substrates are dislocated across bilayer, in cytosol substrates are polyubiquinated

Question 5

Golgi

Answer 5

• Stack maturation model
  1. Vesicular transport model
• Vesicles bud off, fuse to cisternae membranes moves from cisternae to next
• Budding vesicles can be used to transport molecules back to ER
• Cisternae stationary

2. Cisternal maturation model
   - Golgi apparatus = ↑ dynamic
   - Cis cisternae move forards
   - Vesicles formed but only transport molecules back
   - Evidence = cis Golgi expressed w/ GFP, trans red GFP

Question 6

Exocytosis

Answer 6

a) Constitutive exocytosis (all cells, secretes components of EM, important in transporting proteins like receptors)
b) Regulated exocytosis (neutransmitted release: vesicles fuse w/ plasma membrane, digestive E release)

Question 7

Endocytosis

Answer 7

• Basic mechanism = specific molecule binds a receptor on surface → membrane pinches → coated vesicle
• Experiment w/ HRP
• w/ EGF uptake = ↑ efficient

Question 8

Endosome

Answer 8

• Membrane-bound compartment
  1. Early endoscope (primary sorting station, different compartments, ligands + receptors sorted separately, ligands concentrated in a tubular compartment)

2. Late endosome (normally round, manage receptors x recycled, fuse w/ lysosome, membrane x digested)

Question 9

Lysosome

Answer 9

• Acidic due to action of VTPases
• CHloride channels neutralise H+
• Contains hydrolyses

Question 10

Autophagy

Answer 10

• Cell removes unnecessary components, merges
• Delivers cytoplasmic components to vacuole
• Eliminates waste
• Recycling resources when scarce

Question 11

Eukaryotic end-membrane system overview

Answer 11

• Cargo sorting in donor → membrane deformed → vesicle buds → vesicle transport → recognition

Question 12

Cargo sorting

Answer 12

• pH
• Membrane thickness/hydrophobic mismatch
• Sorting at the ER (what remains vs goes further, CopII ER → Golgi)
• Sorting at Cis-Golgi (KDEL, binding + release = pH dependent, KKxx motif, binding of cargo to copI Δ activity of CopI
• sorting at trans-Golgi (endoscope + lysosome, constitutive exocytosis, regulated exocytosis, mannose-6-phosphate receptor)
• sorting for autophagy (mitophagy, PINKI)

Question 13

Membrane bending

Answer 13

• Planar polarisation of bilayer
• Sealed compartment
• Cytoskeletal attachment
• Membrane-binding protein (reticular, BAR-domain protein)
• Types of coat (Clathrin, copI, copII, caveolae)

Question 14

Membrane fusion + fission

Answer 14

• Surface of lipid bilayer ↑ hydrated
• Dynamin = GTPase, constricts point, buds off
• SNARES (V, t, need to re-use, protein threaded through NSAD pore)

Question 15

ESCRT

Answer 15

• ESCRTO recognises late endosome
• ESCRTI recruited
• ESCRTIII assembles as spiral, causes overbidding
• Only known mechanism

Question 16

Regulation

Why?

Answer 16

• Avoid futile cycles
• Maintain membrane identity
• Maintain organelle integrity

Question 17

Cargo-dependent trafficking

Answer 17

Receptor-mediated endocytosis

• Drug receptors on PM, exocytosed
• theory behind drug tolerance
• AP complex recruit Cathrin coat
• Cargo receptor for COPI + II

Question 18

Phospholipid

Answer 18

• PIP3 has phosph. at different positions on head
• G coupled receptor activates phospholipase PIP2 → DAG + PIP3
• PIP2 → PI4P
• PIP2 necessary for recruitment of adaptors

Question 19

GTPase

Answer 19

• GEF GDP → GTP
• After membrane fusion, GTP recycled
• ARF-GTP exposes hydrophobic patch
• ARF -GAP binds curved membranes
• RAB GTpase, attach to membrane, ↑ diverse effect