Free clathrin triskelions are required for the stability of clathrin-associated adaptor protein (AP-2) coated pit nucleation sites

Abstract

In this study image correlation spectroscopy was used to demonstrate the presence of two populations of clathrin in situ, on intact cells. In the periphery of the cell ~35% of the clathrin triskelions are free within the cytosol while ~65% are in large aggregates, presumably coated pits. Although endocytosis is inhibited at low temperature, free clathrin triskelions are still present and small AP-2 aggregates (of ~20 proteins), or coated pit nucleation sites, are still observed. Following hypertonic treatment, or cytoplasmic acidification, free clathrin triskelions within the cytosol are depleted and all of the clathrin becomes associated with the membrane. Under these conditions coated pit associated AP-2 remains while the smaller AP-2 aggregates, or coated pit nucleation sites, dissociate. This indicates that the stabilization of AP-2 coated pit nucleation sites requires the presence of free clathrin triskelions within the cytosol. Furthermore, this indicates that free clathrin is required for the early stages of coated pit formation and presumably the continuation of the clathrin-mediated endocytic process. We also provide indirect evidence that AP-2 binding to the membrane in coated pit nucleation sites may be regulated in part by binding to internalization-competent membrane receptors.Key words: adaptor protein (AP-2), clathrin, distribution, nucleation sites, endocytosis.