Endocytosis assays in intact and permeabilized cells.

Clathrin-coated pits and vesicles represent the major ports of entry into most eukaryotic cells. As well as performing housekeeping functions (e.g.

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation.

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment.

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase.

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs.