Clathrin-Mediated Endoytosis

Volker Haucke, Michael Krauß, Haucke, Volker, Krauß, Michael

Clathrin-mediated endocytosis (CME) is a universal and evolutionarily conserved process that enables the internalization of numerous cargo proteins, including receptors for nutrients and signaling molecules, as well as synaptic vesicle reformation. Multiple genetic and chemical approaches have been developed to interfere with this process. However, many of these tools do not selectively block CME, for example by targeting components shared with clathrin-independent endocytosis pathways or by interfering with other cellular processes that indirectly affect CME.Clathrin, via interactions of endocytic proteins with its terminal domain (TD), serves as a central interaction hub for coat assembly in CME. Here, we describe an ELISA-based, high-throughput screening method used to identify small molecules that inhibit these interactions. In addition, we provide protocols for the purification of recombinant protein domains used for screening, e.g., the clathrin TD and the amphiphysin B/C domain. The screen has been applied successfully in the past, and ultimately led to the discovery of the Pitstop® family of inhibitors, but remains in use to evaluate the inhibitory potency of derivatives of these compounds, and to screen for completely novel inhibitor families.