title: Amphiphysin I is associated with coated endocytic intermediates and undergoes stimulation-dependent dephosphorylation in nerve terminals.
abstract: Amphiphysin I is an abundant presynaptic protein that interacts via its COOH-terminal src homology 3 (SH3) domain with the GTPase dynamin I and the inositol-5-phosphatase synaptojanin. Both dynamin I and synaptojanin I have a putative role in synaptic vesicle recycling and undergo rapid dephosphorylation in rat brain synaptosomes stimulated to secrete by a depolarizing stimulus. We show here that amphiphysin I also undergoes constitutive phosphorylation and stimulationdependent dephosphorylation. Dephosphorylation of amphiphysin I requires extracellular Ca2+ and is unaffected by pretreatment of synaptosomes with tetanus toxin. Thus, Ca2+ influx, but not synaptic vesicle exocytosis, is required for dephosphorylation. Dephosphorylation of amphiphysin I, like dephosphorylation of dynamin I and synaptojanin I, is inhibited by cyclosporin A and FK-506 (0.5 microM), two drugs that specifically block the Ca2+/calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 microM), which blocks protein phosphatases 1 and 2B. We also show by immunogold electron microscopy immunocytochemistry that amphiphysin I is localized in the nerve terminal cytomatrix and is partially associated with endocytic intermediates. These include the clathrin-coated buds and dynamin-coated tubules, which accumulate in nerve terminal membranes incubated in the presence of guanosine 5'-3-O-(thio)triphosphate. These data support the hypothesis that amphiphysin I, dynamin I, and synaptojanin I are physiological partners in some step(s) of synaptic vesicle endocytosis. We hypothesize that the parallel Ca2+-dependent calcineurin-dependent dephosphorylation of amphiphysin I and of its two major binding proteins is part of a process that primes the nerve terminal for endocytosis in response to a burst of exocytosis.
authors.affiliation: Department of Cell Biology and the Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: R
authors.initials: R
authors.lastname: Bauerfeind
authors.forename: K
authors.initials: K
authors.lastname: Takei
authors.forename: P
authors.initials: P
authors.lastname: De Camilli
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; C075637:amphiphysin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C097702:synaptojanin; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D010739:Phospholipase D; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D034281:Dynamins