A requirement for ankyrin binding to clathrin during coated pit budding

Recent studies suggest that the mobility of clathrin-coated pits at the cel l surface are restricted by an actin cytoskeleton and that there is an obli gate reduction in the amount of spectrin on membranes during coated pit bud ding. The spectrin-actin cytoskeleton associates with membranes primarily t hrough ankyrins, which interact with the cytoplasmic region of numerous int egral membrane proteins. We now report that the fourth repeat domain (D4) o f ankyrin, binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin -coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LD L). Ankyrin, peptides that contained repeat domains other than D4 had no ef fect on either in vitro budding or internalization of LDL. Finally, immunof luorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-coated pits during endocytosis.