M&R 6.2 Receptor-mediated endocytosis

Question 1

Describe phagocytosis

Answer 1

Internalisation of material by a (specialised) cell (e.g. macrophages & neutrophils)

Membrane EVAGINATES and particle is internalised by a membrane zippering mechanism

Clears damaged cellular material and invading organisms for destruction

Question 2

Describe pinocytosis

Answer 2

INVAGINATION of plasma membrane to form a lipid vesicle

Permits uptake of impermeable extracellular SOLUTES and retrieval of plasma membrane

2 types: Fluid phase & Receptor-mediated endocytosis (RME)

Question 3

What is receptor-mediated endocytosis (RME)?

Answer 3

Selective internalisation of molecules into a cell via binding to specific cell surface receptors

Question 4

Describe the structure of LDL cholesterol and how this facilitates its transport into cells that need it

Answer 4

Core of esterified cholesterol ester
Covered by phospholipid & cholesterol monolayer containing Apoprotein B

Animal cells that need cholesterol synthesis LDL receptors which specifically recognises ApoB
LDL particle internalised and delivered to lysosomes –> cholesterol released from cholesterol esters

Question 5

Where are the LDL receptors localised on the membrane? (for RME)

Answer 5

In clathrin-coated pits

Question 6

Describe what happens when LDL binds to LDL receptors in the clathrin-coated pits, up until LDL dissociates from the receptor

Answer 6

Coated pit invaginates and pinches off, forming a coated vesicle
Vesicles are uncoated (via ATP-dependent uncoating)
Uncoated vesicle fuses with endosome/CURL (compartment for uncoupling of receptor and ligand)
pH in endosome is maintained low (5.5-6) relative to the cytoplasm (7.2)
At the lower pH the receptor has less affinity for the LDL particle so they dissociated

Question 7

Describe how the dissocated LDL receptors and LDL are sorted in the endosome

Answer 7

The receptors are sequestered to an area of endosome membrane that buds off and recycles the LDL receptor to the plasma membrane

The endosomes containing LDL fuse with lysosomes, so cholesterol can be hydrolysed from the cholesterol esters and released into the cell

Question 8

Describe the structure of a clathrin coat

Answer 8

Clathrin forms 3-legged triskelions (which each contain 3 clathrin chains (the ‘legs’) and 3 light chains which orient them

The triskelions associate to form a basket-like structure made of hexagons & pentagons

Question 9

Do clathrin coats require energy to form?

Answer 9

No - they form spontaneously.

(It is thought that clathrin is attached to the cytosolic side of the membrane. Clathrin molecules start to form cages spontaneously which drives the invagination of membrane & pinching off as a coated vesicle)

Question 10

Does vesicle uncoating require energy?

Answer 10

Yes

the assembly is spontaneous so the uncoating must be energy-driven

Question 11

How are clathrin-coated vesicles uncoated?

Answer 11

ATP-dependent uncoating protein binds and stabilises the freed coat proteins

Clathrin triskelions are recycled back to newly-forming clathrin coated pits

Question 12

Overall, what happens to the ligand and what happens to the receptor in LDL endocytosis?

Answer 12

Ligand degraded

Receptor recycled

Question 13

Describe 3 types of LDL receptor mutations that have been found in homozygous individuals with hyperchoelsterolaemia

Answer 13

1. Non-functional receptor (associated with pits so internalised as normal, but has low affinity for LDL so doesn’t bring LDL with it)
2. Receptor deficiency (mutation prevents expression of LDL receptor)
3. Receptor binding normal (receptor can bind LDL normally but is missing cytoplasmic domain which interacts with clathrin coat. Therefore LDL receptors distributed over whole cell surface - not localised over pits so not internalised)

Question 14

Describe the process of RME of Fe3+ up until fusion with the endosome

Answer 14

2 x Fe3+ ions bind to apotransferrin in the circulation = transferrin
Transferrin binds to the transferrin receptor in the clathrin-coated pits (apotransferrin cannot bind to this receptor)
Transferrin internalised in a coated vesicle, then uncoated and fuses with the endosome

Question 15

In RME of Fe3+, describe what happens after fusion with the endosome

Answer 15

Receptor with transferrin (apotransferrin + 2xFe3+) attached enters acidic endosome
Fe3+ ions dissociate, leaving apotransferrin
Apotransferrin remains attached to the transferrin receptor because of the low pH
Apotransferrin-receptor complex is sorted for recycling back to plasma membrane
Neutral pH at plasma membrane allows apotransferrin to dissociate from transferring receptor

Question 16

in RME of Fe3+, what is the fate of the ligand and the fate of the receptor?

Answer 16

Ligand recycled (apotransferrin)
Receptor recycled (transferrin receptor)

Question 17

In RME of insulin, are the receptors always localised over the coated pits?

Answer 17

No - the insulin receptor only congregates over the coated pits when insulin is bound
(binding probably induces a conformational change in the receptor which allows it to be recognised by the coated pit)

Question 18

In RME of insulin, what happens after the uncoated vesicle fuses with the endosome?

What is the advantage of this?

Answer 18

Insulin remains bound to the insulin receptor in the endosome
The entire insulin-receptor complex is targeted to the lysosome for degradation

This allows down-regulation of number of insulin receptors on the cell surface in response to high circulating insulin levels -> desensitises cell

Question 19

In RME of insulin what is the fate of the ligand and what is the fate of the receptor?

Answer 19

Ligand degraded

Receptor degraded

Question 20

What is transcytosis?

Answer 20

A process by which macromolecules are uptaken into vesicles via RME, then transported across the cell and ejected from the opposite cell membrane via exocytosis

Question 21

What is the purpose of transcytosis?

Answer 21

To transport large molecules across cells

Question 22

Name some molecules which are transported by transcytosis, and where they are transported between

Answer 22

Maternal immunoglobulins transported from mother to fetus via placenta

Immunoglobulin A (IgA) transported from the circulation into bile (in the liver)

Question 23

Describe the process of transcytosis in relation to ligands and receptors

Answer 23

Ligand binds to receptor, uptaken as a coated vesicle, uncoated & fuses with endosome
Ligand remains bound to receptor at endosome pH
Transfer vesicle transports the ligand-receptor complex across cell
During transport the receptor is cleaved - results in release of ligand with bound ‘secretory component’ - part of original receptor

Question 24

In transcytosis, what is the fate of the ligand and the fate of the receptor?

Answer 24

Ligand transported

Receptor transported

Question 25

Explain how membrane-enveloped viruses can take advantage of RME

Answer 25

They can bind to cells via fortuitous association with receptors in the clathrin-coated pits - this tricks cells into internalising them by RME
Then enter endosome and use pH to alter their membrane and reveal membrane fusion domains
This allows viral membrane to fuse with endosome membrane and release viral RNA into cytoplasm
Then uses host cell machinery to replicate RNA & proteins in order to bud new viruses at the cell membrane (using membrane of invaded cell to envelop new virus particles)

Question 26

How do cholera toxin and diptheria toxin enter cells?

Answer 26

Both bind GM1 ganglioside (a phospholipid-like structure found in the plasma membrane) then enter cell via RME