The N terminus of amphiphysin II mediates dimerization and plasma membranetargeting

Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 do mains that interact with dynamin and synaptojanin. The amphiphysins may fun ction in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions wit h clathrin and AP2, We have recently identified and cloned several amphiphy sin II splice variants that differentially incorporate clathrin-binding dom ains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphys in II variants to clathrin-coated pits on plasma membranes purified from tr ansfected COS-7 cells. Full-length amphiphysin II targets to the plasma mem brane where it partially co-localizes with clathrin. However, splice varian ts and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not af fect amphiphysin II distribution. Surprisingly, plasma membrane targeting w as dependent on the presence of a 31-amino acid alternatively spliced seque nce at the N terminus of amphiphysin II, a result confirmed using subcellul ar fractionation, In binding assays, the 31-amino acid sequence was also fo und to facilitate amphiphysin dimerization mediated through the N terminus, Taken together, these data support a role for the N terminus of amphiphysi n II in membrane targeting during endocytosis.