Lecture 13 - Endocytosis

Question 1

What are the principles of endocytosis?

Answer 1

Deform the membrane.

Include or Exclude Proteins/Lipids in the ‘budding’ compartment (terminology: include or exclude ‘cargo’).

‘Budding’ of the compartment from the plasma membrane.

Uncoat the vesicle (if necessary) and deliver (fuse) the cargo to another compartment.

Question 2

How is the membrane deformed?

Answer 2

Amphipathic helices: Polar and hydrophobic amino acids on opposite sides of the helix
Loop insertion: Loops with hydrophobic amino acids
Classic lattices: Lattices bind cargo proteins but the lattice forms a curved polymer
BAR domain proteins: Proteins bind the bilayer via a curved surface e.g BAR domain containing proteins

Question 3

How is cargo included/excluded?

Answer 3

Clathrin binds huge amount of accessory proteins. Clathrin binds AP-2, and AP-2 subsequently binds lipids
Lots of accessory proteins are then binding the cargo these transmembrane proteins.
There are proteins in the plasma membrane which are binding AP-2 and clathrin, clathrin is binding to AP-2 and clathrin and AP-2 are binding a huge amount of accessory proteins. Dense protein mesh

Question 4

Describe cargo adaptors

Answer 4

Cargo Adaptors are the proteins that link cargo into the clathrin-coated pits. AP-2 is the adaptor protein at the plasma membrane

Appendage domains bind accessory proteins
The trunk domains bind to membranes and cargo
The hinge regions bind to clathrin

Question 5

Describe how AP-2 binds plasma membrane lipids

Answer 5

Lipid binding (4 x PI(4,5,)P2 per AP-2) allows cargo binding following a conformational change
AP-2 binds plasma membrane lipids and then proteins
First of all AP-2 is in a locked conformation. It then binds PI(4,5)P2, inducing an open conformation and releasing binding pockets so then it can bind those acidic patch dileucines and tyrosine motifs. Cargo receptor bound to AP2

Question 6

Describe the structure of the clathrin coat

Answer 6

Clathrin triskelions consists of 3 heavy and 3 light chains which assemble to form a clathrin cage around a membrane
(hexagons and pentagons – like a football)

Question 7

Describe clathrin-mediated endocytosis

Answer 7

Receptor is activated by ligand binding
Activated receptor causes binding to adaptor protein AP2
AP2 then binds clathrin
Clathrin then forms its cage
Vesicle is formed
Vesicle uncoating takes place. Naked transport vesicle can go on to early endosome or Golgi, use its SNAREs to fuse and transport molecules from one membrane to another

Question 8

Describe how budding and vesicle scission takes place

Answer 8

Dynamin

• hexameric GTPase
• forms a ring around the neck of the budding vesicle
• uses GTP hydrolysis to achieve fission

Question 9

Describe how vesicle uncoating and delivery of cargo takes place

Answer 9

Association of receptor with clathrin coated pit
Endocytosis
Uncoating
Fusion with early endosome (EE)
and then either:
Maturation to late endosome
(near Golgi, more acidic than EE and different Rabs to EE)
and Fusing with/trafficking to lysosome for degradation
or:
Recycling to plasma membrane via transport vesicles that bud from the EE and can fuse to form recycling endosomes

Question 10

What are the possible fates of receptors?

Answer 10

1. The receptor recycles between an intracellular compartment and the plasma membrane e.g. LDL-receptor, Transferrin receptor
2. Transcytosis e.g. Fc-receptor carrying antibodies from apical to basolateral side of the cell
3. Degradation e.g. Epidermal Growth Factor Receptor in the Lysosome

Question 11

Describe LDL receptor recycling

Answer 11

Inherited LDL receptor trafficking disorders:

Familial hypercholesterolemia
(high serum cholesterol)

LDL receptors are present on the membrane but do not associate with clathrin coated pits

Other receptors are trafficked normally

One of two defects:
mutation of NPXY motif
mutation of AP2 adaptor

Question 12

Describe EGF receptor degradation

Answer 12

EGF-R only associates with clathrin coated pits upon ligand binding (fast endocytosis) – unoccupied receptor is distributed evenly on plasma membrane (and is also endocytosed – but very slowly)
EGF-R, like LDL-R has a tetrapeptide internalisation sequence (YXXF, also found in transferrin receptor), which interacts with AP2 complex)

Internalisation also involves the tyrosine kinase activity of the receptor, which initiates signalling too.

Most EGF receptors do not recycle but are degraded in the lysosome – leading to receptor AND ligand down-regulation/degradation
EGF-R, like LDL-R has a tetrapeptide internalisation sequence (YXXF, also found in transferrin receptor), which interacts with AP2 complex)

Internalisation also involves the tyrosine kinase activity of the receptor, which initiates signalling too.