Identification and functional characterization ofArabidopsisAP180, a binding partner of plant αC-adaptin

Abstract

Clathrin-mediated endocytosis is a well-studied uptake mechanism for nutrients and signalling receptors in mammalian cells that depends on the coordinated interaction of coat proteins and endocytic network proteins to perform the internalization. In this process AP180 promotes the assembly of clathrin triskelia into coated membrane patches at the plasma membrane, while α-adaptin interacts with various network proteins that are in turn required for the budding of the coated pits. The process of clathrin-mediated endocytosis in plants has not been dissected at the molecular level, nor have the members of an analogous uptake machinery been functionally described. In this respect, we have investigated the AP180 and α-adaptin orthologs from Arabidopsis thaliana: At-AP180 and At-αC-Ad. Both plant proteins display the same structural features as their mammalian counterparts and fulfill the same basic functions. To identify their interacting partners, the ear region of At-αC-Ad and the C-terminal region of At-AP180 were used as fusion proteins in pull-down experiments and plasmon-resonance measurements. At-αC-Ad binds several mammalian endocytic proteins, and its interaction with At-AP180 requires the DPF motif. At-AP180 functions as a clathrin assembly protein that promotes the formation of cages with an almost uniform size distribution. Deletion of the single DLL motif abolished the assembly activity of At-AP180 almost completely, but did not affect its binding to triskelia, suggesting the existence of additional binding determinants.