Vesicular Transport

Question 1

How is exit from the RER controlled by chaperones?

Answer 1

Chaperones bind to misfolded proteins in RER lumen which prevent them from leaving
Normally folded proteins are able to bud off
If misfolded proteins do not refold normally, they are transported back to cytosol where they are degraded (proteosome or trafficked to lysosomal pathways) or they are degraded in ER lumen by ubiquitin dependent degradation.
- highly selective process
- exit signals involved
- quality control - ensures correctly folded proteins leave

Question 2

Where do proteins go after golgi apparatus?

Answer 2

• Lysosome
• Plasma membrane
• Sent back to ER (if they have ER retention signal, KDEL)

go back because otherwise one directional flow would lead to depletion of ER lipids and ER resident proteins

go back with retrieval signals: luminal proteins, KDEL and membrane proteins, KKXX

release of proteins from golgi is pH dependent: charge properties of protein change -> protein interaction changes -> can release/pick up cargo

GOLGI = LOW pH
ER = neutral pH

Question 3

What experiments led to the hypothesis of trafficking?

Answer 3

1960s - G.Palade
Pulse chase experiment: used radioactive proteins made in ER, trafficked where they were
Later improved by J.Lippinscott Shwartz
Used green fluorescent proteins (GLP) fused to end of chosen protein (viral glycoprotein - as glycolysation and other post translation modifications through secretory pathways therefore protein can be tracked at these different stages)
and tracked fluorescent signal over time
used mutant version where glycoprotein retained in golgi at certain temperatures and released at certain temp -> to see conc of protein released from golgi

Question 4

Describe the basic transport by budding and fission

Answer 4

1. Coat protein assembly at membrane forces bilayer to begin to bend
2. As they gather at the membrane, coat proteins may also select cargo to be packaged into vesicle
3. More coat protein binding results in formation of sphere of membrane
4. Once coated, vesicle pinches off, coat is detached and cargo filled vesicle is transported to destination

Question 5

Describe COPI and COPII vesicle coat

Answer 5

COPI surrounds vesicles originating from CIS GOLGI to ER

• Formed of 7 polypeptides (no inner and outer)
• Arf1 GTPase similar to Sar1 from COPII
• activated by GEF to convert GDP to GTP and initiate membrane distortion and curvature of golgi membrane

COPII surrounds vesicles from ER to CIS GOLGI

• contains inner coat (sec23/sec24), outer coat (sec13/sec31) and sec12 (contains Sar1)
• shapes vesicle
• cargo sorting
• associates with switch (GTPase) so that assembly is reversible

Sar1 = GTP- Binding protein
- Uses chemical energy from GDP to cause
conformational change → induces a strain and membrane deformation of ER membrane → initiate budding process

Vesicle formation is triggered by Sec12 when it exchanges Sar1-GDP → Sar1-GTP

Sec24 responsible for binding to cargo (recognises cargo using DXE)

inner coat serves as platform for outer coat polymerisation (sec13/31 complex) → form a cuboctohedron

• flexible enough to transport large cargos
• rigid enough to impact curvature on ER membrane

Question 6

How are snares responsible for vesicle fusion and ensuring specificity?

Answer 6

Snares are proteins which help to overcome the unfavourable event of vesicle fusion
Snares are proteins that perform 2 major functions:

1. Snares Promote Fusion
   V snare and T snare form a snare pin which exerts a force between donor and receptor vesicle which brings two membranes together for fusion
   - many neurotoxins work by inhibiting Snare complex formation
2. Snares Ensure Specificity of membrane fusion

Different v/t snare complexes form at different steps of intra cellular transport

Question 7

How are snares recycled?

Answer 7

NSF (N-ethylmaleimide Sensitive Factor) and a-SNAP are proteins essential in unwinding and dissociating V and T snare helical complex and recycling them

• requires energy from ATP hydrolysis to power ‘spring loaded’ motor to unwind snare pins apart

a-SNAP and cis snare complex recognition based on shape and electrostatic recognition, not lock and key

• allows a-SNAP to recognise and recycle diversity of snare complexes that can be formed