IDENTIFICATION OF THE MAJOR SYNAPTOJANIN-BINDING PROTEINS IN BRAIN

Synaptojanin is a nerve-terminal enriched inositol 5-phosphatase thoug ht to function in synaptic vesicle endocytosis, in part through intera ctions with the Src homology 3 domain of amphiphysin. We have used syn aptojanin purified from Sf9 cells after baculovirus mediated expressio n in overlay assays to identify two major synaptojanin-binding protein s in rat brain. The first, at 125 kDa, is amphiphysin. The second, at 40 kDa, is the major synaptojanin-binding protein detected, is highly enriched in brain, is concentrated in a soluble synaptic fraction, and co-immunoprecipitates with synaptojanin. The 40-kDa protein does not bind to a synaptojanin construct lacking the proline-rich C terminus, suggesting that its interaction with synaptojanin is mediated through an Src homology 3 domain. The 40-kDa synaptojanin-binding protein was partially purified from rat brain cytosol through a three-step procedu re involving ammonium sulfate precipitation, sucrose density gradient centrifugation, and DEAE ion-exchange chromatography. Peptide sequence analysis identified the 40-kDa protein as SH3P4, a member of a novel family of Src homology 3 domain-containing proteins. These data sugges t an important role for SH3P4 in synaptic vesicle endocytosis.