Clathrin/AP2 Flashcards
Which study looked into Clathrin and AP2?
Ter Haar et al (2000)
What is the structure of Clathrin?
a triskelion (3 legged structure) made up of 3x180kDa heavy chains and 3x30kDa light chains. Terminal domains is a beta-propeller with 7 blades formed by beta-sheets attached to an alpha helical linker region.
What is the structure of AP2?
AP2 has a clathrin box motif (unfolded peptide) on its hinge region. It lodges between blades 1 and 2 on terminal domain. 2D to 3D
What is CME?
Clathrin mediated endocytosis is a form of vesicle transport that facilitates the internalization and recycling of receptors
What do clathrin molecules do?
spontaneously assembles into cages at pH 6-6.5 in the presence of mg and ca ions, but in the context of a cell, they polymerize around the cytoplasmic face of an invaginated membrane and act as a reinforced mould in which the membrane vesicle may form
Once the vesicle separates from the membrane, the clathrin cage rapidly dissociates
Clathrin terminal domains bind to AP2 clathrin box motifs
What is the structure and function of terminal domains?
beta-propeller domain which is formed by beta-sheets - has 7 domains/blades
attached to an alpha helical linker region.
How does the terminal domain bind to the clathrin box?
Clathrin box motif is a peptide on the hinge region of AP2.
It lodges between blades 1 and 2 of the beta-propeller structure
What do clathrin box motifs look like?
A peptide containing hydrophobic, acidic residues; lots of L and D residues.
What are the four binding sites on the terminal domain?
- Clathrin box
- Royle box - predicted using sequence analysis then determined via by Royle in 2015
- W box
- Arrestin box
CryoEM contributions to clathrin
CryoEM structure of clathrin cages was solved by Fotin et al 2004, to 8 angstroms resolution which enabled secondary structures to be seen. Crystal structures fitted in.
What did Fotin find?
- a helical tripod at cage vertex where the three heavy chain legs come together in tight bond.
- light chains run along edges but they were mobile so densities not all accounted for
How did crystallography complement function of clathrin?
Wilbur et al 2010 revealed that light chains have a role in the assembly of the cage structure and prevention of aggregation/unproductive structures
What is Hsc70’s role in clathrin disassembly?
Hsc70 and auxilin disassemble the cage. Hsc70 covers clathrin and disrupts interactions. It binds to a binding site underneath the vertex. Requires ATP. EM maps showed low resolution conformational changes in light chains upon binding.
What does AP2 look like?
mickey mouse. A heterotetramer, half alpha, half beta 2 with sigma and mu subunits.
The alpha and beta2 domains each have two binding sites for other adaptor proteins.
Beta2 hinge has clathrin binding motif
