Section 7B: The clathrin and COP coats

Question 1

What is the function of a coat?

Answer 1

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

Question 2

______ scaffolds the shape of the budding vesicle

Answer 2

Clathrin

Question 3

Clathrin forms a structure called…

Answer 3

Triskelions

Question 4

Hexagons and pentagons convex to form a basket called…

Answer 4

Clathrin coated pit

• different sizes represent different stages of bud formation

Question 5

What is the problem with Clathrin?

Answer 5

it does not have the ability to bind membranes on its own

Question 6

Adaptor protein (AP)

Answer 6

• a peripheral membrane protein
• can associate directly with the lipid bilayer
• binds clathrin
• binds cargo: picks what will make it into the vesicle

Question 7

What does the adaptor protein (AP) make the clathrin do?

Answer 7

makes sure that the scaffold or assembly only happens on the surface of the right donor membrane compartment

Question 8

What does the adaptor protein (AP) have to be able to tell?

Answer 8

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

Question 9

Adaptor protein complexes (AP) are intermediates between cargo molecules and clathrin

Answer 9

• 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

Question 10

Is clathrin located on the inside or outside of the cell?

Answer 10

inside; the budding that clathrin does is inward from the plasma membrane

Question 11

The stages of clathrin coat assembly

Answer 11

1. adaptor protein lands
2. recruits clathrin: this will begin process of bending the membrane inwards
3. vesicle formation: eventually, clathrin assembles spontaneously into a sphere
4. concentration of protein in the middle of the sphere

Question 12

Membrane scission of clathrin-mediated vesiculation

Answer 12

• dynamin spiral squeezes neck of the vesicle from the donor membrane

Question 13

What is Dynamin?

Answer 13

• 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

Question 14

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…

Answer 14

then they can’t transmit neurotransmitters to the muscle

Question 15

What controls where and when coat proteins assemble to form a vesicle?

Answer 15

1. Phosphoinositides:
   - controls clathrin coat assembly
2. Specific GTPases:
   - controls COP coat assembly
   - Arf: controls COPI
   - Sar: controls COPII

Question 16

What is the major difference of the phospholipid Phosphatidylinositol (PIP2)?

Answer 16

The sugar Inositol headgroup has 6 hydroxyl groups (6 carbon sugar)
- rest are the same: glycerol backbone, 2 fatty acid tails, a phosphate

Question 17

Why is the 6 hydroxyl headgroup important in Phosphatidylinositol (PIP2) important?

Answer 17

The lipid headgroup can become phosphorylated or modified with phosphate groups on specific positions (4 and 5)

Question 18

Where does phosphorylation occur on Phosphatidylinositol (PIP2)?

Answer 18

Occurs on the part of the lipid that is already polar

Question 19

Phosphatidylinositol (PIP2)

Answer 19

• 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

Question 20

What are Phosphoinositides?

Answer 20

They are Phosphatidylinositol (PIP2) phosphorylated at any position(s)

Question 21

Phosphatidylinositol-4,5-biphosphate

Answer 21

Phosphatidylinositol (PIP2) phosphorylated at 4 and 5 positions of inositol headgroup; controls clathrin coat assembly

Question 22

What does Phosphatidylinositol-4,5-biphosphate regulate?

Answer 22

• 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

Question 23

What membrane is PIP2 found on?

Answer 23

• inner leaflet of plasma membrane

Question 24

Why do clathrin coats form selectively on the plasma membrane?

Answer 24

because the plasma membrane is the only one that has PIP2

Question 25

PIP2 recruit proteins and the headgroup sticks out on which part of the membrane?

Answer 25

the cytosolic face

Question 26

How does AP or AP2 bind to membranes?

Answer 26

It has a unique groove on its surface to bind to the 2 lipid

Question 27

What is the Adaptor Protein (AP) controlled by?

Answer 27

- 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

Question 28

What allows the progression to a couple AP molecules for assembly of the first part of the clathrin coat?

Answer 28

PIP2

Question 29

What causes clathrin coat disassembly?

Answer 29

PIP2 is removed or degraded in the vesicle once it is released from the donor compartment which causes disassembly

Question 30

How does PIP2 control the "opening" of AP2

Answer 30

- 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

Question 31

What is different about the COPII coat?

Answer 31

- doesn't use clathrin - doesn't use AP - doesn't use dynamin - is not regulated by PIP2

Question 32

What is the similar end product of COP and COPII?

Answer 32

the ability to package proteins into a vesicle; in this case from the ER

Question 33

Mechanism of COPII coat formation

Answer 33

1. GTPases 2. Sar1 (GTPase) and Sar1-GEF 3. Sec23/24 and Sec 13/31 (4). Membrane is deformed and buds off from ER

Question 34

Sar1 GTPase regulates assembly of COPII coat on the ER membrane

Answer 34

- 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

Question 35

What type of cargo goes into a COPII coated vesicle or a vesicle made by COPII?

Answer 35

any proteins and lipids that the ER makes; wide range of different types of proteins and lipids

Question 36

GTPase is on and off when...

Answer 36

ON: when it's GTP bound OFF: when it's GDP bound

Question 37

What causes GTPase to turn ON?

Answer 37

the binding of GTP caused by a GEF

Question 38

What causes GTPase to turn OFF?

Answer 38

the binding of GDP caused by GAP

Question 39

What would trigger turning the coat ON controlled by SAR?

Answer 39

the formation of this coat will be triggered by the binding of GTP caused by GEF

Question 40

Where does the hydrophobic antenna go when Sar1 is GTP bound?

Answer 40

it will go and reach into the lipid bilayer

Question 41

What is the breakdown of the first step that causes the COPII coat to begin to form?

Answer 41

1. Sar1-GEF displaces GDP from inactive, cytosolic Sar1-GDP 2. GTP loads onto Sar1 3. Sar1-GTP exposes amphipathic helix (antenna) 4. Sar1-GTP binds strongly to ER membrane

Question 42

Sec23/24

Answer 42

- 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

Question 43

Sec13/31

Answer 43

- 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