Identification of the Major Synaptojanin-binding Proteins in Brain

doi:10.1074/jbc.272.13.8710

Identification of the Major Synaptojanin-binding Proteins in Brain*

From the Department of Neurology and Neurosurgery, Montreal Neurological Institute and the
^‡ Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B4, Canada

^¶ Alfred P. Sloan Research Fellow. To whom correspondence should be addressed:
CBET Group, Montreal Neurological Inst., McGill University, 3801 University Ave., Montreal, PQ H3A 2B4, Canada
. Tel.: 514-398-7355; Fax: 514-398-8106; E-mail: mcpm{at}musica.mcgill.ca

Abstract

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.

Footnotes

↵^§ Medical Research Council of Canada Scholars.
↵* This work was supported by Grant MT-12046 (to W. S. S.) and Grant MT-13461 (to P. S. M.) from the Medical Research Council of Canada and by a Fonds De La Recherche En Santé Du Québec Establishment Grant (to P. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SH3

Src homology 3

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride.
- Received September 11, 1996.
- Revision received November 22, 1996.