support of the Markey Fellowship, to develop new cell free assays, using perforated A431 cells that faithfully reconstitute these events.
We have also developed stage specific biochemical assays that allow us to detect sequential intermediates in endocytic-coated vesicle formation. These assays follow the receptor-mediated endocytosis of transferrin, which is biotinylated with a cleavable disulfide bond (referred to as BSSTfn). The “sequestration” of receptor-bound BSSTfn into an intermediate in vesicle formation, called a constricted coated pit, can be detected when the ligand becomes inaccessible to avidin. However, because constricted coated pits remain accessible to small molecules through a narrow opening at their necks that connects them to the plasma membrane, the BSSTfn sequestered in them can be cleaved by the small membrane-impermeant, reducing agent, MesNa. Thus, late events in vesicle formation, namely the release of a sealed vesicle from the plasma membrane are detected when the BSSTfn becomes resistant to MesNa. Finally, we can focus on measuring the earliest events in vesicle formation, by supplementing assays performed in the presence of limiting concentrations of cytosol with purified coat proteins. The coat protein-stimulated sequestration of BSSTfn into constricted coated pits reflects de novo coat assembly.
Many years of work helped us to biochemically characterize these stages of vesicle formation. We found that while the coat proteins, clathrin and AP2, are necessary for coated vesicle formation, they are not sufficient. For example, intermediate and late events in this process are regulated by the GTPase dynamin. Most of my work in the past 6 years has focused on understanding the function in vivo and in vitro of this GTPase, although it is not subject of the talk today. These late events also require ATP hydrolysis, by an as-yet-unidentified ATPase, as well as numerous accessory proteins that have been implicated by virtue of their interaction with dynamin and with the coat proteins.
Early events in CCV formation require the lipid, phosphatidylinositol-4,5 -bisphosphate (PI-4,5-P2), which provides at least part of the targeting signal for recruitment of AP2 complexes and other endocytic accessory proteins, including dynamin to the plasma membrane. Early events also require ATP hydrolysis and again, we have not identified the ATPase. One candidate, however, will be the subject of my talk today. The role of numerous accessory proteins that interact with adapters and clathrin are not known.
I will focus on the regulation of endocytic vesicle formation. An extremely talented postdoc, Sean Conner, did all of the work I am going to tell you about today. Because the adaptors are a key factor in initiating coat assembly—“the brains of the operation”—we focused our attention on these. The AP2 complex consists of four subunits, called alpha, beta,