Identification of the Major Synaptojanin-binding Proteins in Brain
1997; Elsevier BV; Volume: 272; Issue: 13 Linguagem: Inglês
10.1074/jbc.272.13.8710
ISSN1083-351X
AutoresElaine de Heuvel, Alexander W. Bell, Antoine R. Ramjaun, Kenny K. Wong, Wayne S. Sossin, Peter S. McPherson,
Tópico(s)Biochemical and Structural Characterization
ResumoSynaptojanin 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. 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. INTRODUCTIONSynaptic vesicles are specialized organelles that neurons use to secrete nonpeptide neurotransmitters. Following neurotransmitter release, synaptic vesicle membranes are retrieved by a process thought to involve clathrin-coated pits and vesicles (1McPherson P.S. De Camilli P Semin. Neurosci. 1994; 6: 137-147Crossref Scopus (14) Google Scholar, 2Baverfeind R. David C. Galli T. McPherson P.S. Takei K. De Camilli P. Protein Kinesis: The Dynamics of Protein Trafficking and Stability. Vol. LX. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995: 397-404Google Scholar), and recent data suggest that this endocytic mechanism is active at both high and low rates of exocytosis (3Ryan T.A. Smith S.J. Reuter H. Proc. Natl. Acad. Sci. 1996; 93: 5567-5571Crossref PubMed Scopus (202) Google Scholar). We have identified a 145-kDa protein, referred to as synaptojanin, which is enriched in nerve terminals and appears to function in synaptic vesicle endocytosis (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar, 5McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Synaptojanin is an inositol 5-phosphatase that dephosphorylates inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and phosphatidylinositol 4,5-bisphosphate at the 5′ position of the inositol ring (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Inositol phosphate metabolism has been implicated in a variety of membrane trafficking events including endocytosis (7De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (659) Google Scholar). In addition, synaptojanin has an N-terminal domain that is homologous to the cytosolic domain of the yeast SacI protein. SacI mutants show genetic interactions with actin as well as with the yeast secretory mutants sec6, sec9, and sec14 (8Novick P. Osmond B.C. Botstein D. Genetics. 1989; 121: 659-674Crossref PubMed Google Scholar, 9Cleves A.E. Novick P.J. Bankaitis V.A. J. Cell Biol. 1989; 109: 2939-2950Crossref PubMed Scopus (189) Google Scholar), and more recently SacIp has been demonstrated to mediate ATP transport into the yeast endoplasmic reticulum (10Mayinger P. Bankaitis V.A. Meyer D.I. J. Cell Biol. 1995; 131: 1377-1386Crossref PubMed Scopus (58) Google Scholar).Synaptojanin was initially identified based on its ability to bind to the Src homology 3 (SH3) 1The abbreviations used are: SH3Src homology 3PCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisPVDFpolyvinylidene difluoride. domains of Grb2 (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar). Cloning of synaptojanin revealed a 250-amino acid proline-rich domain at its C terminus (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar) that contains at least five sequences forming potential SH3 domain-binding sites (11Mayer B.J. Eck M.J. Curr. Biol. 1995; 5: 364-367Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). A second, 170-kDa isoform of synaptojanin is present in a wide variety of tissues including neonate brain but is not detected in adult brain (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar, 12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The 170-kDa synaptojanin isoform is generated by alternative splicing of the synaptojanin gene leading to the presence of an additional 266-amino acid proline-rich domain with at least three additional SH3 domain-binding consensus sequences as compared with the 145-kDa isoform (12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).Synaptojanin also binds to the SH3 domain of amphiphysin (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Amphiphysin was first identified in chicken synaptic fractions (13Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar) and mammalian amphiphysin, which is concentrated in presynaptic nerve terminals, has been implicated in synaptic vesicle endocytosis (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). A role for amphiphysin in endocytosis is supported by studies on its yeast homologues, RVS 161 and RVS 167 (15David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar, 16Crouzet M. Urdaci M. Dulau L. Aigle M Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 17Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). Mutations in these genes cause an endocytosis defect characterized in part by an impairment in α-factor receptor internalization (18Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar). Further, amphiphysin is known to interact with AP2 (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 19Wang L.-H. Sudhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), a component of the plasma membrane clathrin coat (20Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (304) Google Scholar). Evidence of a role for the SH3 domain of amphiphysin in synaptic vesicle endocytosis is provided by its interaction with dynamin. A role for dynamin in endocytosis was first determined based on its identity with the gene product of the Drosphila shibire mutant (21Chen M.S. Obar R.A. Schroeder C.C. Austin T.W. Poodry C.A. Wadsworth S.C. Vallee R.B. Nature. 1991; 351: 583-586Crossref PubMed Scopus (435) Google Scholar, 22van der Bliek A.M. Meyerowitz E.M. Nature. 1991; 351: 411-414Crossref PubMed Scopus (588) Google Scholar). Mutations in Drosophila dynamin leads to a block in synaptic vesicle endocytosis (23Kosaka T. Ikeda K. J. Neurobiol. 1983; 14: 207-225Crossref PubMed Scopus (252) Google Scholar), and recent data suggest that dynamin functions in the nerve terminal by mediating the fission of endocytic vesicles (24Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (650) Google Scholar, 25Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (654) Google Scholar). Thus, it appears likely that SH3 domain-mediated interactions of amphiphysin with synaptojanin are important to the endocytic function of synaptojanin in vivo. SH3 domain interactions involving Grb2 have also been recently demonstrated to be important for clathrin-mediated endocytosis of the epidermal growth factor receptor in non-neuronal cells (26Wang Z. Moran M.F. Science. 1996; 272: 1935-1939Crossref PubMed Scopus (146) Google Scholar). Specifically, disruption of Grb2 interactions with the epidermal growth factor receptor blocks receptor endocytosis, and epidermal growth factor can stimulate a transient association of Grb2 with dynamin (26Wang Z. Moran M.F. Science. 1996; 272: 1935-1939Crossref PubMed Scopus (146) Google Scholar). Thus, SH3 domain-mediated interactions appear to function widely in clathrin-mediated endocytosis.Here, we have used purified synaptojanin in overlay assays to identify its preferred binding targets in brain. In addition to amphiphysin, we identified a 40-kDa synaptojanin-binding protein that is highly enriched in brain, is concentrated in soluble synaptic fractions, and co-immunoprecipitates with synaptojanin. Purification and peptide sequence analysis revealed the 40-kDa protein as SH3P4, a novel SH3 domain-containing protein that was identified from a mouse library screened with a Src SH3 ligand peptide (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (213) Google Scholar). SH3P4, along with SH3P8 and SH3P13, define a family of similar proteins of unknown function (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (213) Google Scholar). Our data strongly implicate SH3P4, and perhaps other family members, in synaptic vesicle endocytosis.DISCUSSIONSynaptojanin is an inositol 5-phosphatase implicated in synaptic vesicle endocytosis (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar, 5McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar, 14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). Synaptojanin was initially isolated based on its ability to bind to the SH3 domains of Grb2 (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar), and a role for Grb2 in endocytosis was recently demonstrated (26Wang Z. Moran M.F. Science. 1996; 272: 1935-1939Crossref PubMed Scopus (146) Google Scholar). Synaptojanin also binds to the SH3 domain of amphiphysin (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar, 14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar), and several pieces of evidence implicate amphiphysin in endocytosis, including its SH3 domain-dependent interaction with dynamin (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). Thus, it appears that SH3 domain-mediated interactions play a general role in endocytosis. In an effort to better characterize the nature of SH3 domain-mediated protein-protein interactions with synaptojanin, we generated synaptojanin and a synaptojanin deletion construct in Sf9 cells using the baculovirus system. The proteins were then purified on nickel-agarose using a His6 tag engineered into the N terminus of the constructs. To characterize the baculovirus expressed synaptojanin, we compared the affinity of amphiphysin binding to synaptojanin versus dynamin. When amphiphysin or Grb2 are used as substrates for the purification of SH3 domain-binding proteins from brain extracts, greater amounts of dynamin than synaptojanin are isolated (5McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar), likely owing to higher levels of dynamin expression in brain. However, as shown here, when equal amounts of purified dynamin and synaptojanin are analyzed, amphiphysin shows stronger binding to synaptojanin than dynamin. It has been proposed (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar) that amphiphysin may serve to target dynamin to sites of synaptic vesicle endocytosis via its dual interactions with AP2 (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 19Wang L.-H. Sudhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and dynamin. Amphiphysin may also play a role in targeting synaptojanin to endocytic sites. The higher affinity of synaptojanin than dynamin for amphiphysin binding may be important to allow for synaptojanin targeting in the presence of high dynamin concentrations in the nerve terminal.We used synaptojanin purified from Sf9 cells in a gel overlay assay to identify two major synaptojanin-binding proteins with molecular masses of approximately 125 and 40 kDa. The 125-kDa synaptojanin-binding protein was identified as amphiphysin based on its co-migration with amphiphysin on SDS-PAGE and its precipitation with amphiphysin antibodies. The identification of amphiphysin as a major synaptojanin-binding protein strongly suggests that the assay is effective in identifying relevant synaptojanin-binding partners in vitro and further suggests that amphiphysin and the 40-kDa protein are the major synaptojanin-binding proteins in vivo.Further characterization of the 40-kDa synaptojanin-binding protein demonstrates that it is highly concentrated in brain and is predominantly a soluble protein that is enriched in cytosol isolated from lysed synaptosomes. Proteins that function in clathrin-mediated endocytosis are often expressed at levels 10-50-fold higher in neuronal versus non-neuronal cells (33Morris S.A. Schmid S.L. Curr. Biol. 1995; 5: 1-3Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). For example, both dynamin and synaptojanin are highly expressed in neurons, whereas these proteins or related isoforms are expressed at lower levels in non-neuronal cells (12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 34Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar, 35Sontag J.-M. Fyske E.M. Ushkaryov Y. Liu J.-P. Robinson P.J. Südhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 36Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (161) Google Scholar). The 40-kDa synaptojanin-binding protein is concentrated in brain but is also detected in testis, a tissue with little or no expression of the 145-kDa isoform of synaptojanin (12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, the testis does express the 170-kDa synaptojanin isoform (12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and this protein also binds strongly to the 40-kDa synaptojanin-binding protein (data not shown). An important role for the 40-kDa synaptojanin-binding protein is also supported by the observation that it co-immunoprecipitates with synaptojanin from rat brain cytosol. This is in contrast to amphiphysin, which does not co-immunoprecipitate with synaptojanin (Fig. 6). The reason for the lack of amphiphysin/synaptojanin co-immunoprecipitation is unclear, but it may be due to a technical reason such as steric interference of the synaptojanin antibody with the site of amphiphysin binding. A more interesting explanation may be that the binding of synaptojanin to the 40-kDa synaptojanin-binding protein excludes amphiphysin binding. Thus, it is possible that the 40-kDa synaptojanin-binding protein could regulate the ability of synaptojanin to bind to amphiphysin, and this could play a key role in regulating the targeting of synaptojanin to sites of endocytosis.To identify the 40-kDa protein, we purified it from rat brain cytosol and subjected it to peptide sequence analysis. The sequence analysis identifies the 40-kDa synaptojanin-binding protein as SH3P4, a novel SH3 domain-containing protein with a predicted molecular mass of 39,880 Da (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (213) Google Scholar). The identification of the 40-kDa synaptojanin-binding protein as an SH3 domain-containing protein is consistent with our observation that the 40-kDa protein does not bind to a synaptojanin deletion construct lacking the proline-rich C terminus. Further, the predicted isoelectric point of 5.3 for SH3P4 (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (213) Google Scholar) is consistent with its elution from the DEAE ion exchange column in high salt. SH3P4, which was identified from a mouse library screened with a Src SH3 ligand peptide, is 75 and 63% identical to SH3P8 and SH3P13, respectively, two other proteins identified in the same screen (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (213) Google Scholar). These three proteins define a novel protein family of unknown function. Our data strongly implicate SH3P4, and perhaps other family members, in synaptic vesicle endocytosis. It will be of interest to determine if the interaction of SH3P4 can regulate the ability of synaptojanin to bind to amphiphysin and thus regulate the targeting of synaptojanin to sites of endocytosis. INTRODUCTIONSynaptic vesicles are specialized organelles that neurons use to secrete nonpeptide neurotransmitters. Following neurotransmitter release, synaptic vesicle membranes are retrieved by a process thought to involve clathrin-coated pits and vesicles (1McPherson P.S. De Camilli P Semin. Neurosci. 1994; 6: 137-147Crossref Scopus (14) Google Scholar, 2Baverfeind R. David C. Galli T. McPherson P.S. Takei K. De Camilli P. Protein Kinesis: The Dynamics of Protein Trafficking and Stability. Vol. LX. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995: 397-404Google Scholar), and recent data suggest that this endocytic mechanism is active at both high and low rates of exocytosis (3Ryan T.A. Smith S.J. Reuter H. Proc. Natl. Acad. Sci. 1996; 93: 5567-5571Crossref PubMed Scopus (202) Google Scholar). We have identified a 145-kDa protein, referred to as synaptojanin, which is enriched in nerve terminals and appears to function in synaptic vesicle endocytosis (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar, 5McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Synaptojanin is an inositol 5-phosphatase that dephosphorylates inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and phosphatidylinositol 4,5-bisphosphate at the 5′ position of the inositol ring (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Inositol phosphate metabolism has been implicated in a variety of membrane trafficking events including endocytosis (7De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (659) Google Scholar). In addition, synaptojanin has an N-terminal domain that is homologous to the cytosolic domain of the yeast SacI protein. SacI mutants show genetic interactions with actin as well as with the yeast secretory mutants sec6, sec9, and sec14 (8Novick P. Osmond B.C. Botstein D. Genetics. 1989; 121: 659-674Crossref PubMed Google Scholar, 9Cleves A.E. Novick P.J. Bankaitis V.A. J. Cell Biol. 1989; 109: 2939-2950Crossref PubMed Scopus (189) Google Scholar), and more recently SacIp has been demonstrated to mediate ATP transport into the yeast endoplasmic reticulum (10Mayinger P. Bankaitis V.A. Meyer D.I. J. Cell Biol. 1995; 131: 1377-1386Crossref PubMed Scopus (58) Google Scholar).Synaptojanin was initially identified based on its ability to bind to the Src homology 3 (SH3) 1The abbreviations used are: SH3Src homology 3PCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisPVDFpolyvinylidene difluoride. domains of Grb2 (4McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (149) Google Scholar). Cloning of synaptojanin revealed a 250-amino acid proline-rich domain at its C terminus (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar) that contains at least five sequences forming potential SH3 domain-binding sites (11Mayer B.J. Eck M.J. Curr. Biol. 1995; 5: 364-367Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). A second, 170-kDa isoform of synaptojanin is present in a wide variety of tissues including neonate brain but is not detected in adult brain (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar, 12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The 170-kDa synaptojanin isoform is generated by alternative splicing of the synaptojanin gene leading to the presence of an additional 266-amino acid proline-rich domain with at least three additional SH3 domain-binding consensus sequences as compared with the 145-kDa isoform (12Ramjaun A.R. McPherson P.S. J. Biol. Chem. 1996; 271: 24856-24861Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).Synaptojanin also binds to the SH3 domain of amphiphysin (6McPherson P.S. Garcia E. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Amphiphysin was first identified in chicken synaptic fractions (13Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar) and mammalian amphiphysin, which is concentrated in presynaptic nerve terminals, has been implicated in synaptic vesicle endocytosis (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). A role for amphiphysin in endocytosis is supported by studies on its yeast homologues, RVS 161 and RVS 167 (15David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar, 16Crouzet M. Urdaci M. Dulau L. Aigle M Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 17Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). Mutations in these genes cause an endocytosis defect characterized in part by an impairment in α-factor receptor internalization (18Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar). Further, amphiphysin is known to interact with AP2 (14David C. McPherson P.S. Mundigal O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 19Wang L.-H. Sudhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), a component of the plasma membrane clathrin coat (20Robinson M.S. Curr. Opin. 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Our data strongly implicate SH3P4, and perhaps other family members, in synaptic vesicle endocytosis.
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