Lecture 26

Question 1

What is clathrin-mediated endocytosis?

Answer 1

A process of particle internalization occurring at clathrin-coated “pits” in the plasma membrane

Question 2

What does clatrhin-mediated endocytosis use to trigger the internalization event?

Answer 2

Uses ligand/receptor

Question 3

What are ligands?

Answer 3

Ligands are usually growth factors or other soluble factors (proteins, molecules)
Receptor/ligand complexes are concentrated into patches on the membrane
Then internalized into vesicles

Question 4

Does clathrin work on its own?

Answer 4

Clathrin does not work on its own
It uses an assortment of proteins to form the vesicle and release the clathrin lattice once the particle is internalized

Question 5

What are some proteins that work with clathrin to make a “clathrin coated pit”?

Answer 5

FCHo proteins
Adaptor protein-2 (AP2) (aka assembly protein-2)
Epsin
EPS15
Amphiphysin
Dynamin and Actin are used in the final steps of vesicle formation
Once the particle is internalized clathrin must be released
It uses Hsc70 and Auxillin

Question 6

How is membrane curvature initiated?

Answer 6

Membrane curvature is initiated by FCHo1/2

Question 7

What is FCHo1/2?

Answer 7

These are BAR (Bin-Amphiphysin-Rys) proteins with very slight curvature
They bind directly to the plasma membrane
Recruit scaffold proteins (EPS15) often through SH3 domains that bind proline-rich regions of other proteins
Once initiated, other BAR proteins with increased curvature are recruited to the developing pit

Question 8

What are 2 mechanisms used by BAR proteins to curve membrane?

Answer 8

1) The natural curvature of the BAR proteins
2) Wedges are inserted into the plasma membrane lipids only in the leaflet of the membrane of BAR protein docking

Question 9

What is the Adaptor protein-2 (AP-2)?

Answer 9

A 4 subunit complex (a, b2, u2, o2)
The numbers refer to the “2” in AP-2 (there are 4 different AP’s 1,2,3,4)
The subunits are called “adaptins”

Question 10

What is alpha adaptin?

Answer 10

Large sized
Binds to PI (4,5)P2 (AKA: PIP2, part of lipids) in the plasma membrane

Question 11

What is o2 Adaptin?

Answer 11

Small sized
Binds to transmembrane receptors

Question 12

What is beta2 Adaptin?

Answer 12

Large sized
Binds directly to clathrin
Promotes clathrin assembly

Question 13

What is u2 Adaptin?

Answer 13

Medium sized
Interacts with tyrosine-based sorting signals of transmembrane receptors

Question 14

What does Epsin do?

Answer 14

Helps coordinate clathrin coat formation
Can deform the membrane on its own
Can cause liposomes for form into tubes on its own (meaning it can curve membrane and make tubes)
Discovered through its binding to EPS15

Question 15

How many domains does Epsin have?

Answer 15

4 domains

Question 16

What are the 4 domains of Epsin?

Answer 16

ENTH (epsin N-terminal homology): bind to membrane and can also recruit clathrin to the membrane. A domain found in many proteins. Always at the N-terminus of the protein
UIM: Ubiquitin interacting motifs
Clathin/AP-2 binding domain
EH: Epsin Homology Domain

Question 17

What is EPS15?

Answer 17

Epidermal grow factor receptor pathway substrate 15
Originally discovered as a phosphorylation substrate of the epidermal growth factors (EGF) receptor kinase
Binds to the alpha-subunit of AP2
Binds and acts independetly of EGF

Question 18

What area in the AP-2 binding domain is proline rich and which is ubiquitously interacted with?

Answer 18

P= proline rich region
C= ubiquitously interaction

Question 19

Is actin needed for CME?

Answer 19

Actin is crucial for CME
It is driven through the recruitment of the GTPase Adc42 and N-WASp
Actin polymerization occurs at the plasma membrane surface and pushes the vesicle

Question 20

How do Auxilin and Hsc70 come into play?

Answer 20

When the clathrin coated vesicle has formed, the clathrin cage must be disassembled to release the vesicle
This is accomplished through the ATPase Hsc70 and Auxilin
They destabilize the attachments of clathrin, AP2 and other coat proteins for un-coating

Question 21

What mechanism are tight junction proteins endocytosed?

Answer 21

CME: can remove claudins, occludin and JAM from the plasma membrane of epithelial cells
Caveolin: used to remove claudin-5, occludin and JAM from the plasma membrane of endothelial cells

Question 22

What happens once the proteins are endocytosed?

Answer 22

ACCUMULATE IN EARLY ENDOSOMES
This has been shown for Cl1, Cl5, Cl16, Occludin, JAM, Zo1 in epithelial cells and endothelial cells
Causes decreased barrier function

Question 23

What happens if you use siRNA against one of the early endosomal markers (Rab5)?

Answer 23

If you use siRNA against one of the early endoomal markers (Rab5), you get increase Cl1 at the plasma membrane in endothelial cells
Now, where can these proteins go?

Question 24

What 4 places can these proteins go?

Answer 24

1) Recycling pathway
2) Degradation pathway
3) “Bulk-flow” recycling pathways (use of narrow tubes with large surface area to recycle many things at once)
4) To the trans-Golgi network

Question 25

What happens with Occludin, Cl1 and Cl2 in fibroblasts and epithelial cells?

Answer 25

Can undergo continuous recycling (endocytosis and recycling back to the plasma membrane)

Question 26

Where else have occludin and Cl1 been found?

Answer 26

Occludin and Cl1 have also been seen in the degradation pathway, associated with UBIQUITIN E3 ligases

Question 27

Look at video on how clathrin works

Answer 27

As bacteria comes in, actin will make hole in plasma membrane bigger (pushing bacteria in)
Dynamin will pinch it off