Integrating protein copy numbers with interaction networks to quantify stoichiometry in clathrin-mediated endocytosis

Abstract

Abstract Proteins that drive processes like clathrin-mediated endocytosis (CME) are expressed at copy numbers within a cell and across cell types varying from hundreds (e.g. auxilin) to millions (e.g. clathrin). These variations contain important information about function, but without integration with the interaction network, they cannot capture how supply and demand for each protein depends on binding to shared and distinct partners. Here we construct the interface-resolved network of 82 proteins involved in CME and establish a metric, a stoichiometric balance ratio (SBR), that quantifies whether each protein in the network has an abundance that is sub- or super-stoichiometric dependent on the global competition for binding. We find that highly abundant proteins (like clathrin) are super-stoichiometric, but that not all super-stoichiometric proteins are highly abundant, across three cell populations (HeLa, fibroblast, and neuronal synaptosomes). Most strikingly, within all cells there is significant competition to bind shared sites on clathrin and the central AP-2 adaptor by other adaptor proteins, resulting in most being in excess supply. Our network and systematic analysis, including response to perturbations of network components, show how competition for shared binding sites results in functionally similar proteins having widely varying stoichiometries, due to variations in both abundance and their unique network of binding partners.