Amphiphysin I is associated with coated endocytic intermediates and undergoes stimulation-dependent dephosphorylation in nerve terminals

Rudolf Bauerfeind, Kohji Takei, Pietro De Camilli

Research output: Contribution to journalArticle

135 Citations (Scopus)

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 stimulation-dependent 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 μM), two drugs that specifically block the Ca2+/calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 μM), 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.

Original languageEnglish
Pages (from-to)30984-30992
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number49
DOIs
Publication statusPublished - Dec 5 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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