The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting.
Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 domains that interact with dynamin and synaptojanin. The amphiphysins may function in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions with clathrin and AP2. We have recently identified and cloned several amphiphysin II splice variants that differentially incorporate clathrin-binding domains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphysin II variants to clathrin-coated pits on plasma membranes purified from transfected COS-7 cells. Full-length amphiphysin II targets to the plasma membrane where it partially co-localizes with clathrin. However, splice variants and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not affect amphiphysin II distribution. Surprisingly, plasma membrane targeting was dependent on the presence of a 31-amino acid alternatively spliced sequence at the N terminus of amphiphysin II, a result confirmed using subcellular fractionation. In binding assays, the 31-amino acid sequence was also found to facilitate amphiphysin dimerization mediated through the N terminus. Taken together, these data support a role for the N terminus of amphiphysin II in membrane targeting during endocytosis.
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
A R
AR
Ramjaun
J
J
Philie
E
E
de Heuvel
P S
PS
McPherson
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D019281:Dimerization; D004705:Endocytosis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics
D002966:Clathrin; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C075637:amphiphysin