Vesicular Transport Flashcards
lecs 14,15,16: vesicular transport, membrane contact sites and adaptor proteins
1
Q
What are the different types of coated vesicles and what do they do?
A
- Clathrin coated vesicles (TGN to lysosomes/ bud from cell surface)
- COP1 vesicles (important for froward and retrograde transport through the golgi)
- COP2 vesicles (ER to Golgi complex)
2
Q
What are the essential components for all transport vesicle formation?
A
- GTPase
- Adaptor proteins
- Coat
3
Q
What do adaptor proteins do?
A
Recognises signals in cargo and ensures they get packaged into a coated vesicle by linking them to the coat
4
Q
How do small GTPases work?
A
- Molecular switch
- Exist in a inactive GDP form
- Conversion of inactive to active requires exchange from GDP to GTP mediated by GEFs
- GTP to GDP catalysed by GAP (GTPase activating proteins) which help GTP hydrolysis
5
Q
How is a protein coat like that on Cop II coated vesicles formed?
A
The recruitment of cytosolic components onto a specific membrane, these membrane proteins can cycle on and off the vesicle
6
Q
What are small GTPases used for in terms of transport vesicle formation?
A
In their active form, they are important for the recruitment of adaptor proteins
7
Q
What are GEFs and GAPs?
A
GEF = Determines the amount of active GTPase there is in a cell (regulates GDP-> GTP)
GAPs= GTPase activating proteins which help hydrolyse GTP ( regulates GTP-> GDP)
The balance between these two depends on how active the G protein is in general, v. important!
8
Q
How are GTPases seen in vesicular transport into the nucleus (RAN)
A
- Ran is in its GDP form outside the nucleus
- Cargo binds to nuclear receptors and enters
- GEF is localised to chromatin and transfers RanGDP to RanGTP
- GTP binds to the cargo-receptor complex, cargo dissociates due to conformational change and stays in the nucleus
- RAN GTP exists nucleus where it encounters GAP which hydrolyses RAN forming RAN GDP which no longer binds to the nuclear receptors
- The GEF being in the nucleus ensures directionality
9
Q
What are the specific components for COP II?
A
GTPase: Sar 1
Adaptor: Sec23/24
Coat: Sec 13/31
(Liz says dw too much ab the numbers just remember its a Sec complex and the categories)
10
Q
How is a COPII vesicle formed?
A
- GEF for SAR1 in membrane exchanges GDP for active GTP complex allowing recruitment of adaptor complex, Sec 23 unit binds to Sar1
- Sec 24 segment of the adaptor recognises the signal on the cargo receptor which allows it to be incorporated into the COPII vesicle
- The actual vesicle is formed by budding of donor membrane
- When the bud is completely formed it buds off to form a coated vesicle
- It is quite packed inside the bud to try and avoid the escaping of proteins
11
Q
What do coat proteins provide?
A
A structural scaffold
12
Q
Describe centrifugation to get ER membranes (used for in vitro reconstitution of COPII)
A
- Isolate ER membranes through homogenisation and centrifugation
- The centrifugation tube as an increasing gradient of sucrose conc.
- The rough ER at bottom (high sucrose conc.) the smooth ER float at top (low sucrose conc.)
- Rough ER extracted with needle
- By selecting ER membranes with specifically labelled proteins and vesicles with specifically labelled cargo proteins you can then run this separate rough ER through a sucrose gradient and observe what makes up the ER as the vesicles and ER will separate at different bands
13
Q
What was shown in the reconstitution assay regarding what was needed to make a COPII vesicle?
A
- Sar 1
- Sec 23/24
- Sec 13/31 (the coat)
- ATP
-GTP
Sar 1 and Sec23/24 are recruited from the cytosol
14
Q
What does the SNARE in the vesicle do?
A
- Target the vesicle to acceptor compartment
- Uncoating of vesicle needs to occur for fusion
- Accumulation can occur if uncoating doesn’t happen
15
Q
What can the adaptor protein also act as on COPII?
A
- A GAP for Sar 1 (aka turns it off)
- This GAP activity is enhanced when coat is recruited meaning uncoating can occur when needed
- Summary: Sar1 is needed in active form to recruit the adaptor and form the coat, once this forms and vesicle buds off the Sec23 acts as a GAP and inactivates Sar1 to disassemble the membrane
16
Q
How are looking at mutant GTPases good experimentally?
A
- You can see which stage of trafficking doesn’t occur when certain GTPases are mutant
- E.g. expression of Sar1 in its GDP form inhibits COPII formation because it cannot recruit the adaptor protein etc.
17
Q
What is AP2?
A
A major clathrin coated vesicle adaptor protein
18
Q
What is the structure of AP2?
A
- Two large subunits: alpha and beta
- Determinant which allows binding to clathrin
- Smaller subunits which recognise cargo
19
Q
Describe the Rab family
A
- GTPases
- Member of the Ras superfamily
- Originally found in the brain with a very specific subcellular location
- 60-70 Rab family members in mammalian cells
- Cycle between membrane and cytoplasm
- Required for fusion
20
Q
What is the main member of the Rab family and why?
A
Rab 5 because it is a major regulator and one that we know lots about
21
Q
When the Rab is activated by RabGEFs what happens?
A
- Rab interacts with effector molecules
- Rab switched off by rabGAPs
22
Q
Where are each form of Rab found?
A
GDP form= cytoplasm
GTP form= membrane
23
Q
Describe the Rab cycle
A
- Rab recruited from cytoplasm in GDP form
- Changes to GTP activated form
- Associated with SNARE proteins
- These complex facilitate the vesicle being targeted to its designated membrane
- Other proteins (tether proteins) that are bound to Rab act as a tether and tether the vesicle to the target membrane helping the anchoring
- After this happens a RabGAP promotes hydrolysis of GTP on Rab changing it back to GDP
- A chaperone (GDI) takes the Rab away protecting the fatty acid part of the Rab from the cytoplasm
24
Q
How do Rabs regulate the movement of cargo?
A
- Rab cascade
- Rabs interact with effectors and contribute to the particular function of an organelle
- To pass on the cargo to the next compartment/ organelle there is a clever mechanism where the effector for Rab(x) acts as an exchange factor (GEF) for the next Rab associated with the next organelle
- Means the cargo moves through the endocytic pathway
25
What is Cranio-lenticulo-sutural dysplasia caused by?
- Sec23A mutation
- Abnormal endoplasmic reticulum to golgi trafficking
26
What does CLSD lead to?
Problems regarding connective tissue
Deformed face
27
How does the mutation appear in immunofluorescence, what does this suggest and what did they find?
The Golgi appears swollen with collagen implying the collagen may not be being trafficked
- Found to be a defect in functional CopII vesicles in the ER in mutants
28
What are liposomes?
'Fake lysosomes' prepared with lipids
29
What did they do with the liposomes (CLSD)?
Added Sec23A either mutant or wild type and added Sar1 in GDP form or GTP form
- Question was can Sec23A bind or is it just recognised
- Found that Sec23A can bind so its not the binding thats the problem
30
What did the following experiment(CLSD) find to be the problem?
The Sec23A adaptor is not good at recruiting the coat which means the vesicle does not form very well
31
If you cant make COPII coated vesicles, why are only some tissues effected in the patients with the disease (CLSD)?
- Other tissues aren't effected because other paralogs are expressed at higher levels and can compensate (??)'
32
Collagen will not fit into a COPII vesicle, how is it packaged?
- Accessory proteins which enable the COPII vesicle to rearrange to allow long fibrous proteins in
- TANGO1 also helps with the size of collagen helping translocation from the ER to Golgi
33
Why are zebrafish good to study?
- Fast generation time
- Can be used for disease screens
34
What are the rough and smooth ER the site of?
Rough ER= Protein synthesis
Smooth ER= Lipid synthesis and calcium storage
35
What are intramembrane contact sites?
Membrane of one organelle is in close contact with the membrane of another organelle
36
What did Bernhard and Roullier discover?
Starvation, hepatectomy or poisoning disrupts the ER contact sites
37
Is there any fusion or membrane insertion at intramembrane contact sites?
No, just strong contact
38
What does Live cell imaging allow us to look at with MCS?
The dynamics of the contact site
e.g. Er tubule wrapping around mitochondria causing fission
39
What is correlative light and electron microscopy?
- Takes a flourescent image and allows you to look at that spot in electron microscopy
- Localisation of flourescently labelled proteins
- Can correlate different colour spots and look at areas in high resolution
40
What is the size of the gap between the contact sites?
10-80nm
41
What is the molecular machinery of MCS?
Tethers: Protein which stems from ER membrane and prevents fusion, can also move molecules e.g. transferring a lipid
42
Explain how calcium mobilisation is a function of MCS?
- The sarcoplasmic reticulum is the ER of muscle cells
- SR is very close to the plasma membrane and has invaginations to increase SA
- In the SR there is a protein called Stim1, when muscle contracts calcium is depleted. This is sensed by Stim1 so it forms an oligomeric structure and migrates to an area of ER tubules where it forms a contact site with the plasma membrane
- Membrane site is rich with Pip2 where the contact stimulates uptake of extracellular calcium and once in the cytosol pumps Ca2+ back into the ER
43
Explain how lipid transfer is a function of MCS?
- Lipids distributed unidirectionally to other organelles using MCS
- Uses concentration gradients
- Tether proteins can act as lipid transfer proteins
- tether proteins have a hydrophobic pore to accommodate lipids and protect them from the aqueous environment
44
What can defects in lipid transfer using MCS lead to?
- Niemann Pick Disease C
- Defects in transferring cholesterol out of the lysosome to other organelles can cause problems
45
How is signalling a function of MCS?
- ER contacts with early and late endosome important in signalling
- MCS good for signalling of epidermal growth factor receptor which moves through endocytic pathway being phosphorylated and dephosphorylated
- Phosphatase is located in the membrane which dephosphorylates EGFR so it can move into the endosome membrane (MCS is therefore v improtant!)
- The endosome may be moving through this cell and needs this contact with the ER to do so
46
How is organelle fission an example of the function of MCS?
ER tubules can wrap around the mitochondria and cause fission
47
What are some diseases linked to Membrane Contact Sites (MCS)?
Neuronal diseases such as ALS and Hereditary Spastic Paraplegia
