Research on various Endocytic Proteins and Membrane Trafficking Pathways
Clathrin-mediated endocytosis is a major pathway for the reformation of synaptic vesicles following exocytosis. By reconstitution of endocytic components we have been defining the functions of individual components of the coat apparatus. The major components of the coat are clathrin, AP2 adaptors and AP180. A number of other proteins (including epsin, amphiphysin and dynamin) are largely excluded from the clathrin-coated vesicles after scission but are nevertheless essential to its formation. Clathrin provides a scaffold and limiting structure for the vesicle. In vitro it has previously been shown to form cage-like structures. The AP2 adaptors link the clathrin lattice to the membrane and to cargo to be incorporated into the forming vesicle. In our studies we have used a combination of structural and biochemical approaches to study the function of AP180, epsin and dynamin in vesicle formation.
The large GTPase dynamin forms a helical collar around the neck of an invaginating clathrin-coated vesicle, where it may regulate, pinch or pop the vesicle from the parent membrane. We have shown that GTP hydrolys
is is coupled to vesicle scission and that on GTP hydrolysis dynamin spirals undergo a length-wise extension in vitro- which we believe drives the vesicle away from the membrane causing lipid fission.
Epsin1 Pages Drives membrane curvature during AP2 clathrin-mediated endocytosis
EpsinR Pages Found on TGN and endosomes. By analogy with epsin1, it is likely involved in driving membrane curvature during clathrin-coated vesicle formation from these compartments.
AP180 promotes clathrin polymerisation into satellites (patches the same size as clathrin-coated vesicles) on membranes containing PtdIns(4,5)P2. These patches of polymerisation are not invaginated.
Amphiphysin is involved in endocytosis in the brain and in T-tubule formation in muscles. It has an N-terminal N-BAR domain (BAR domain shown above). From the structure we were able to show that analogous BAR domains are found in many different protein families, implicating many of these proteins in membrane trafficking and especially in membrane deformation.
Endophilin I is involved in endocytosis in the brain. Amphiphysin N-BAR domain has an N-terminal amphipathic helix that can insert partially into the membrane, but endophilin has two amphipathic helices per monomer and thus its interaction with the membrane in more stable.
BAR domains come in an assortment of curvatures. Thus they can stabilize/induce a spectrum of membrane deformations. While the F-BAR domains have a more shallow concave surface than BAR/N-BAR domains, they are capable of generating various membrane curvatures (seen as tubules of varying diameter). This is due to the variety of angles F-BAR dimers occupy on membranes.
