Phosphorylation of threonine 156 of the mu 2 subunit of the AP2 complex isessential for endocytosis in vitro and in vivo

The clathrin-coated pit is the major port of entry for many receptors and p athogens and is the paradigm for membrane-based sorting events in higher ce lls ill. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesi cles, allowing dissection of the machinery required for sequestration of re ceptors into these structures [2-6], The AP2 adaptor complex is a key eleme nt of this machinery linking receptors to the coat lattice, and it has prev iously been reported that AP2 can be phosphorylated both in vitro and in vi vo [7-10]. However, the physiological significance of this has never been e stablished. Here, we show that phosphorylation of a single threonine residu e (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay an d in living cells.