Receptor-mediated endocytosis plays an Important role in allowing mammalian cells to sense and respond to their environment. During the last ten years, significant progress has been made in understanding this fundamental process, but a number of questions remain unanswered. Continuation of studies of the endocytic system is proposed. These studies will combine fluorescence spectroscopy of living cells, somatic cell genetics and cell-free analysis of endocytic compartments. A new class of endocytosis mutants, those with defects in recycling from endosomes to the plasma membrane, will be isolated by fluorescence-activated cell sorting. Initial members of this class of mutants have been isolated, demonstrating the feasibility of this approach. The characteristics of these mutants will be determined, and the genes responsible for the defect(s) will be isolated. The long term goals of this aspect of the project are to understand the processes of receptor recycling and endosomal fission events at the molecular level. Previous work on this project has demonstrated that the Na,K-ATPase plays a role in regulating endosomal pH in living cells. This phenomenon occurs in some, but not all, cell types. A second goal of this proposal is understanding the means by which the traffic of Na,K-ATPase to (and possibly from) endosomes occurs in various cell types.