Dynamics of Auxilin1 and GAK in clathrin-mediated traffic

Abstract

ABSTRACTClathrin coated vesicles formed at the plasma membrane lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 “uncoating ATPase”. The J-domain containing proteins, auxilin1 (Aux1) and auxilin2/cyclin-G dependent kinase (GAK), recruit Hsc70. Aux1 and GAK are closely related homologs, each with a phosphatase- and tensin-like (PTEN-like) domain, a clathrin-binding region, and a C-terminal J-domain; GAK has an additional, N-terminal Ser/Thr kinase domain. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin mediated membrane scission has released the vesicle from the plasma membrane. We show here that these bursts represent recruitment of a very small number of auxilins such that even 4-6 molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated-pit assembly. We have also shown previously that clathrin coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. We now show that differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.