Identification of the major synaptojanin-binding proteins in brain.
Synaptojanin is a nerve-terminal enriched inositol 5-phosphatase thought to function in synaptic vesicle endocytosis, in part through interactions with the Src homology 3 domain of amphiphysin. We have used synaptojanin purified from Sf9 cells after baculovirus mediated expression in overlay assays to identify two major synaptojanin-binding proteins 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 procedure 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 suggest an important role for SH3P4 in synaptic vesicle endocytosis.
de Heuvel
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E
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
Bell
A W
AW
Ramjaun
A R
AR
Wong
K
K
Sossin
W S
WS
McPherson
P S
PS
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D018411:Spodoptera; D018909:src Homology Domains
D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; C075637:amphiphysin; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases