Section 7B: The clathrin and COP coats Flashcards
What is the function of a coat?
bind to a donor membrane compartment causing a change in shape of the membrane, so that you form a bud, then causing a scission or release of that vesicle from the target protein
______ scaffolds the shape of the budding vesicle
Clathrin
Clathrin forms a structure called…
Triskelions
Hexagons and pentagons convex to form a basket called…
Clathrin coated pit
- different sizes represent different stages of bud formation
What is the problem with Clathrin?
it does not have the ability to bind membranes on its own
Adaptor protein (AP)
- a peripheral membrane protein
- can associate directly with the lipid bilayer
- binds clathrin
- binds cargo: picks what will make it into the vesicle
What does the adaptor protein (AP) make the clathrin do?
makes sure that the scaffold or assembly only happens on the surface of the right donor membrane compartment
What does the adaptor protein (AP) have to be able to tell?
it has to be able to tell what the membrane is binding to; tell the difference between the plasma membrane and the ER because it binds to the plasma membrane not the ER
Adaptor protein complexes (AP) are intermediates between cargo molecules and clathrin
- during clathrin-mediated endocytosis, cargo is sorted and enriched in budding vesicles
- cargo may be membrane proteins or soluble cargo
- if soluble cargo, this is first bound to transmembrane proteins called receptors
Is clathrin located on the inside or outside of the cell?
inside; the budding that clathrin does is inward from the plasma membrane
The stages of clathrin coat assembly
- adaptor protein lands
- recruits clathrin: this will begin process of bending the membrane inwards
- vesicle formation: eventually, clathrin assembles spontaneously into a sphere
- concentration of protein in the middle of the sphere
Membrane scission of clathrin-mediated vesiculation
- dynamin spiral squeezes neck of the vesicle from the donor membrane
What is Dynamin?
- a protein that assembles in a spiral
- a special GTPase that does not require an external GAP
- undergoes a conformational change with the hydrolysis of GTP that cuts the neck of the vesicle from the donor membrane
- the GTPase activity of dynamin is assembly-regulated
- the hydrolysis of GTP to GDP by dynamin causes scission
Some of the vesicles that dynamin works to make are the same vesicles and neurons that hold neurotransmitters, so if you can’t make the vesicles that hold the neurotransmitters…
then they can’t transmit neurotransmitters to the muscle
What controls where and when coat proteins assemble to form a vesicle?
- Phosphoinositides:
- controls clathrin coat assembly - Specific GTPases:
- controls COP coat assembly
- Arf: controls COPI
- Sar: controls COPII
What is the major difference of the phospholipid Phosphatidylinositol (PIP2)?
The sugar Inositol headgroup has 6 hydroxyl groups (6 carbon sugar)
- rest are the same: glycerol backbone, 2 fatty acid tails, a phosphate
Why is the 6 hydroxyl headgroup important in Phosphatidylinositol (PIP2) important?
The lipid headgroup can become phosphorylated or modified with phosphate groups on specific positions (4 and 5)
Where does phosphorylation occur on Phosphatidylinositol (PIP2)?
Occurs on the part of the lipid that is already polar
Phosphatidylinositol (PIP2)
- phospholipid on cytosolic leaflet of bilayer
- inositol headgroup has 6 hydroxyl groups
- several of these hydroxyl groups can be phosphorylated and dephosphorylated
- the phosphorylation of the inositol headgroup is carried out by specific lipid kinases, and dephosphorylation occurs due to specific lipid phosphatases
What are Phosphoinositides?
They are Phosphatidylinositol (PIP2) phosphorylated at any position(s)
Phosphatidylinositol-4,5-biphosphate
Phosphatidylinositol (PIP2) phosphorylated at 4 and 5 positions of inositol headgroup; controls clathrin coat assembly
What does Phosphatidylinositol-4,5-biphosphate regulate?
- they are the signal to start assembling clathrin coated pits
- highly enriched at the plasma membrane
- allows AP2 (adaptor protein 2), dynamin and other proteins to bind membrane
What membrane is PIP2 found on?
- inner leaflet of plasma membrane
Why do clathrin coats form selectively on the plasma membrane?
because the plasma membrane is the only one that has PIP2
PIP2 recruit proteins and the headgroup sticks out on which part of the membrane?
the cytosolic face
How does AP or AP2 bind to membranes?
It has a unique groove on its surface to bind to the 2 lipid
What is the Adaptor Protein (AP) controlled by?
- It is controlled by PIP2
- It’s the ability of the very first AP to bind to the membrane and bind to the cargo at the same time
What allows the progression to a couple AP molecules for assembly of the first part of the clathrin coat?
PIP2
What causes clathrin coat disassembly?
PIP2 is removed or degraded in the vesicle once it is released from the donor compartment which causes disassembly
How does PIP2 control the “opening” of AP2
- AP2 starts off in a locked position, unable to do anything: good thing because we dont want AP binding to Clathrin creating coats everywhere in the cell
- As soon as AP interacts with the lipid PIP2, it activates it and opens it
- Once open it can do two things: bind to cargo, and clathrin
What is different about the COPII coat?
- doesn’t use clathrin
- doesn’t use AP
- doesn’t use dynamin
- is not regulated by PIP2
What is the similar end product of COP and COPII?
the ability to package proteins into a vesicle; in this case from the ER
Mechanism of COPII coat formation
- GTPases
- Sar1 (GTPase) and Sar1-GEF
- Sec23/24 and Sec 13/31
(4). Membrane is deformed and buds off from ER
Sar1 GTPase regulates assembly of COPII coat on the ER membrane
- material goes from the ER to the Golgi; in order to make that journey, the material from the ER has to get packaged into a vesicle which then fuses with the Golgi
- COPII is involved in forward membrane trafficking from ER to Golgi
- it helps to deliver new synthesized proteins and lipids to the Golgi
- first step of biosynthetic sorting process
What type of cargo goes into a COPII coated vesicle or a vesicle made by COPII?
any proteins and lipids that the ER makes; wide range of different types of proteins and lipids
GTPase is on and off when…
ON: when it’s GTP bound
OFF: when it’s GDP bound
What causes GTPase to turn ON?
the binding of GTP caused by a GEF
What causes GTPase to turn OFF?
the binding of GDP caused by GAP
What would trigger turning the coat ON controlled by SAR?
the formation of this coat will be triggered by the binding of GTP caused by GEF
Where does the hydrophobic antenna go when Sar1 is GTP bound?
it will go and reach into the lipid bilayer
What is the breakdown of the first step that causes the COPII coat to begin to form?
- Sar1-GEF displaces GDP from inactive, cytosolic Sar1-GDP
- GTP loads onto Sar1
- Sar1-GTP exposes amphipathic helix (antenna)
- Sar1-GTP binds strongly to ER membrane
Sec23/24
- forms an inner coat that allows the binding to specific cargo molecules for export out of the ER and into Golgi
- only binds to membranes when Sar1-GTP is there
Sec13/31
- COPII subunits form outside coat
- end result: on the surface of the ER because that’s the only place where Sar1 is bound to GTP
