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• W2023981624 abstract "The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca2+-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation. The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca2+-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation. solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor growth hormone glutathioneS-transferase hemagglutinin synaptosome-associated protein of 25 kDa RING-B box coiled-coil polyacrylamide gel electrophoresis Chinese hamster ovary B box C-terminal coiled-coil vesicle-associated membrane protein 2 At synapses, neurotransmitters are released via Ca2+-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. Recent genetic and biochemical studies have revealed that this highly regulated fusion process involves a cascade of protein-protein and protein-lipid interactions (1Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1768) Google Scholar, 2Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (422) Google Scholar). Among them, a ternary protein complex known as the solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)1 complex is of fundamental importance for synaptic vesicle exocytosis (3Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2628) Google Scholar). The SNARE complex is assembled by three neuronal SNAREs, vesicle-associated membrane protein 2 (VAMP2, also called synaptobrevin), and presynaptic plasma membrane proteins syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25). Structural studies have demonstrated that the SNARE complex consists of a parallel four-stranded helical bundle formed by two helices from SNAP-25 and one helix each from VAMP2 and syntaxin 1 (4Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1916) Google Scholar). Interference with the integrity of such a superhelical structure by various mutations in the SNAREs has been shown to inhibit membrane fusion (5Ossig R. Schmitt H.D. de Groot B. Riedel D. Keranen S. Ronne H. Grubmuller H. Jahn R. EMBO J. 2000; 19: 6000-6010Crossref PubMed Scopus (62) Google Scholar, 6Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar). Moreover, specific cleavage of each SNARE by the clostridial neurotoxins prevents the assembly of a stable SNARE complex and blocks neurotransmitter release without affecting the docking of synaptic vesicles (7Hunt J.M. Bommert K. Charlton M.P. Kistner A. Habermann E. Augustine G.J. Betz H. Neuron. 1994; 12: 1269-1279Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 8O'Connor V. Heuss C. De Bello W.M. Dresbach T. Charlton M.P. Hunt J.H. Pellegrini L.L. Hodel A. Burger M.M. Betz H. Augustine G.J. Schafer T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12186-12191Crossref PubMed Scopus (85) Google Scholar). Thus, the formation of the SNARE complex is a crucial event in the synaptic vesicle fusion process. Although the functional importance of the SNARE complex in membrane fusion is well established, its precise role in the fusion process remains unclear. It has been proposed that the formation of the SNARE complex in a trans configuration pulls the apposing membranes into close contact and provides a driving force for membrane fusion (9Hanson P.I. Heuser J.E. Jahn R. Curr. Opin. Neurobiol. 1997; 7: 310-315Crossref PubMed Scopus (334) Google Scholar). Consistent with this view, the assembly of SNARE complexes has been shown to serve as the minimal machinery for membrane fusion in reconstituted liposomes (10Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2015) Google Scholar). Furthermore, the SNARE complex formation in permeabilized PC12 cells is triggered by Ca2+ and coupled directly to exocytosis (11Chen Y.A. Scales S.J. Patel S.M. Doung Y.C. Scheller R.H. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). On the other hand, evidence from studies of yeast vacuole fusion suggests that the SNARE complex does not act at the fusion step (12Ungermann C. Sato K. Wickner W. Nature. 1998; 396: 543-548Crossref PubMed Scopus (279) Google Scholar). Rather, the complex formation occurs at an upstream step to signal other proteins to execute fusion (13Peters C. Mayer A. Nature. 1998; 396: 575-580Crossref PubMed Scopus (325) Google Scholar). In addition, a recent study using synaptosomes suggests that SNARE complexes assemble at the priming step prior to neurotransmitter release and may regulate the amount of synaptic vesicle to undergo exocytosis (14Lonart G. Sudhof T.C. J. Biol. Chem. 2000; 275: 27703-27707Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Whereas the SNAREs and the SNARE complex seem to be universally required for all fusion reactions, synaptic vesicle exocytosis is several orders of magnitude faster and more tightly regulated than any other form of membrane fusion (15Sabatini B.L. Regehr W.G. Nature. 1996; 384: 170-172Crossref PubMed Scopus (315) Google Scholar). To achieve the extraordinary speed, precision, and plasticity of neurotransmission, additional proteins have to be involved to regulate the function of these SNAREs and control temporal and spatial formation of SNARE complexes. In an effort to identify additional proteins that regulate neurotransmitter release, we have performed a search in rat brain for SNAP-25-binding proteins using a yeast two-hybrid screen. We report here the isolation of a novel RING finger protein, termed Spring, that specifically interacts with SNAP-25 and modulates the SNARE complex formation and Ca2+-dependent exocytosis. Yeast two-hybrid screens to identify novel SNAP-25-interacting proteins were performed as described previously (16Chin L.S. Nugent R.D. Raynor M.C. Vavalle J.P. Li L. J. Biol. Chem. 2000; 275: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Prey plasmids from positive clones were rescued and re-transformed into fresh yeast cells with the pPC97-SNAP-25 bait or various control baits to confirm the specificity of the interactions. For cloning of full-length Spring, a partial Spring cDNA probe from the prey clone was used to screen a rat hippocampal cDNA library in λZAPII (Stratagene), according to standard procedures (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The cDNA inserts from positive Spring clones were sequenced multiple times on both strands by an Applied Biosystems 373A DNA sequencer. A polyclonal anti-Spring antibody was raised in rabbit against amino acid residues 138–151 (DDRGLRGFPKNRVL) of Spring. The antibody was affinity-purified using the immunogen peptide coupled to an Aminolink Immobilization column (Pierce). Other antibodies used in this study are as follows: anti-SNAP-25 (SMI 81, Sternberger Monoclonals, Inc.); anti-syntaxin 1 (HPC-1, Sigma); anti-VAMP2 (Wako Pure Chemical Industries, Ltd.); anti-synaptophysin (SVP-38, Sigma); and anti-HA (3F10, Roche Molecular Biochemicals). Northern blot analysis of Spring mRNA expression was performed on a rat Multiple Tissue Northern (MTNTM) blot and a human Multiple Tissue Expression (MTETM) Array (CLONTECH), using a 32P-labeled Spring cDNA fragment from the prey clone as probe (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For Western blot analysis, rat tissues were homogenized in 1% SDS and subjected to SDS-PAGE. The proteins were transferred onto nitrocellulose membranes and probed with anti-Spring and other antibodies. Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence were used to visualize the results. Conventional molecular biological techniques (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were used to generate the constructs in this study. DNA fragments encoding full-length and truncated forms of Spring were subcloned into the following vectors: the pPC97 and pPC86 vectors for yeast two-hybrid interaction studies; the prokaryotic expression vectors pGEX-5X-2 (Amersham Pharmacia Biotech) and pET28c (Novagen) for the production of GST- and His6-tagged fusion proteins; and the mammalian expression vectors pCDNA3.1(+) (Invitrogen) and pCHA (16Chin L.S. Nugent R.D. Raynor M.C. Vavalle J.P. Li L. J. Biol. Chem. 2000; 275: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) for transfection into CHO and PC12 cells. GST or His6-tagged fusion proteins were expressed inEscherichia coli BL21 cells as described previously (16Chin L.S. Nugent R.D. Raynor M.C. Vavalle J.P. Li L. J. Biol. Chem. 2000; 275: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). GST fusion proteins were affinity-purified by using the glutathione-agarose beads (Sigma). His6-tagged proteins were purified using the His·Bind Resin and Buffer kit (Novagen). Protein concentrations were estimated by Coomassie Blue staining of protein bands following SDS-PAGE, using bovine serum albumin as standard. Rat brain extracts were prepared by homogenizing the brains in a homogenization buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 300 mm sucrose, plus protease inhibitors). Triton X-100 was added to the homogenates to a final concentration of 1% and incubated at 4 °C for 30 min. Insoluble material was removed by centrifugation at 100,000 × g for 1 h at 4 °C, and the supernatant was used as the Triton X-100 extract of rat brains. For binding experiments, brain extracts (100 µl) were incubated at 4 °C for 1 h with various GST-Spring fusion proteins immobilized on the glutathione-agarose beads. After extensive washes, bound proteins were eluted by boiling in the Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting. For binding experiments (Figs.4A and 5A), 100 nm GST or various GST-SNARE fusion proteins immobilized on glutathione-agarose beads were incubated with soluble His-Spring (50 nm) in the PBS buffer (140 mm NaCl, 2.7 mm KCl, 10 mmNa2HPO4, 1.8 mmKH2PO4, pH 7.4 plus protease inhibitors) at 4 °C for 3 h under gentle rocking. Bound proteins were analyzed by SDS-PAGE and immunoblotting. For saturation experiments (Fig.4B), 100 nm immobilized GST-SNAP-25 were incubated with increasing amounts of soluble His-Spring or His-syntaxin 1 (residues 1–261) in the PBS buffer and processed as above. The EC50 was defined as the effective concentration of each soluble protein at half-maximal binding. For competition experiments (Fig. 6, A and B), 50 nm immobilized GST-SNAP-25 were incubated with a constant amount of His-Spring (50 nm) and increasing amounts of His-syntaxin 1 or His-VAMP2. To study the effects of the association of Spring with SNAP-25 on the ability of SNAP-25 to interact with other SNAREs (Fig. 7,A–C), immobilized Spring-SNAP-25 binary complexes were assembled by incubation of 100 nm immobilized GST-Spring with His-SNAP-25 (200 nm). After extensive washes to remove unbound SNAP-25, the immobilized Spring-SNAP-25 complexes were incubated with increasing amounts of His-Syntaxin 1, His-VAMP2 or both His-Syntaxin 1 and His-VAMP2 (1:1 molar ratio) in the PBS buffer and processed as above. Control binding experiments were performed by incubation of immobilized GST-SNAP-25 (100 nm) with His-Syntaxin 1, His-VAMP2 or both His-Syntaxin 1 and His-VAMP2 (500 nm each).Figure 5Identification of interacting domains ofSNAP-25and Spring.A, mapping of the Spring binding domain of SNAP-25. Schematic representation of SNAP-25 and its deletion mutants encoded by the GST fusion cDNA constructs is shown on thetop. These fusion proteins were immobilized on glutathione-agarose beads (lower panel, Ponceau S staining) and incubated with His-Spring. Bound Spring was detected by immunoblotting. B, mapping of the SNAP-25-binding domain of Spring. Rat brain homogenate (Input) was incubated with immobilized GST fusion proteins containing full-length or truncated forms of Spring as indicated. Bound SNAP-25 was detected by immunoblotting, and GST-Spring fusion proteins were shown as Ponceau S staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Spring competes with syntaxin 1 andVAMP2for binding toSNAP-25. A, binding competition between Spring and syntaxin 1 to SNAP-25. Immobilized GST-SNAP-25 (50 nm) was incubated with a constant amount of His-Spring (50 nm) and increasing amounts of His-syntaxin 1. Bound Spring and syntaxin 1 were detected by immunoblotting, and GST-SNAP-25 was shown as Ponceau S staining. B, binding competition between Spring and VAMP2 to SNAP-25. Immobilized GST-SNAP-25 (50 nm) was incubated with a constant amount of His-Spring (50 nm) and increasing amounts of His-VAMP. Bound Spring and VAMP2 were detected by immunoblotting, and GST-SNAP-25 was shown as Ponceau S staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Association of Spring withSNAP-25abolishes the ability ofSNAP-25to interact with other SNAREs.A, the association of Spring with SNAP-25 prevents the interaction of SNAP-25 with syntaxin 1. Immobilized Spring-SNAP-25 binary complexes were assembled by incubation of GST-Spring (100 nm) with His-SNAP-25 (200 nm). After extensive washes, the immobilized Spring-SNAP-25 complexes (shown as Ponceau S staining) were incubated with increasing amounts of His-Syntaxin 1. Bound syntaxin 1 was detected by immunoblotting. In thecontrol lane, a parallel binding experiment was performed by incubation of immobilized SNAP-25 (100 nm) with 500 nm His-Syntaxin 1. B, the association of Spring with SNAP-25 prevents the interaction of SNAP-25 with VAMP2. Immobilized Spring-SNAP-25 binary complexes (shown as Ponceau S staining) were assembled as described in A and then incubated with increasing amounts of His-VAMP2. Bound VAMP2 was detected by immunoblotting. In the control lane, a parallel binding experiment was performed by incubation of immobilized SNAP-25 (100 nm) with 500 nmHis-VAMP2. C, the association of Spring with SNAP-25 prevents the assembly of the ternary SNARE complex. Immobilized Spring-SNAP-25 binary complexes (shown as Ponceau S staining) were assembled as described in A and then incubated with increasing amounts of His-syntaxin 1 and His-VAMP2 (1:1 molar ratio). Bound syntaxin 1 and VAMP2 were detected by immunoblotting. In thecontrol lane, a parallel binding experiment was performed by incubation of immobilized SNAP-25 (100 nm) with His-syntaxin 1 and His-VAMP2 (500 nm each).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Subcellular fractionations of rat brain into membrane and cytosol fractions were performed as described (16Chin L.S. Nugent R.D. Raynor M.C. Vavalle J.P. Li L. J. Biol. Chem. 2000; 275: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The membrane fractions were subjected to extraction by 4% Triton X-100, 4 m urea, 1.5 m NaCl, or 100 mm Na2CO3, pH 11.5. For cytoskeleton association studies, rat brains were lysed in a cytoskeleton-stabilizing buffer and separated into a low speed cytoskeleton fraction, a high speed cytoskeleton fraction, and a soluble fraction according to a standard procedure (18Stam J.C. Sander E.E. Michiels F. van Leeuwen F.N. Kain H.E. van der Kammen R.A. Collard J.G. J. Biol. Chem. 1997; 272: 28447-28454Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). For synaptosomal localization studies, rat brain homogenates were fractionated into crude synaptosome fractions as described (19Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (890) Google Scholar). The washed crude synaptosome (P2′) pellet fraction was then fractionated on a three-step Percoll gradient into myelin, mitochondria, and purified synaptosome fractions (20Dunkley P.R. Jarvie P.E. Heath J.W. Kidd G.J. Rostas J.A. Brain Res. 1986; 372: 115-129Crossref PubMed Scopus (388) Google Scholar, 21Wang J.K. Walaas S.I. Sihra T.S. Aderem A. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2253-2256Crossref PubMed Scopus (157) Google Scholar). The purified synaptosome fraction (PG3) was further fractionated into the synaptosomal membranes (LP1), synaptic vesicle (LP2), and cytosol (LS2) fractions (19Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (890) Google Scholar). All protein samples were subjected to SDS-PAGE and immunoblotting. Extracts were prepared from CHO cells transiently transfected with pCHA-Spring and pCDNA3.1-SNAP-25, and immunoprecipitations were performed as described previously (22Chin L.-S. Raynor M.C. Wei X. Chen H. Li L. J. Biol. Chem. 2001; 276: 7069-7078Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), using rat monoclonal anti-HA antibody (3F10) or control rat IgG. For detection of endogenous Spring-SNAP-25 complexes, the clarified supernatant of solubilized P2′ synaptosome fraction was subjected to immunoprecipitation by anti-SNAP-25 antibody (SMI81) or control mouse IgG. The immunocomplexes were recovered by incubation with protein A/G-agarose beads (Santa Cruz Biotechnology) for 1 h at 4 °C. After extensive washes, the immunocomplexes were analyzed by SDS-PAGE and immunoblotting. Exponentially growing PC12 cells were harvested and then cotransfected with 5 µg of pXGH5 encoding human growth hormone and 30 µg of test plasmid as described previously (16Chin L.S. Nugent R.D. Raynor M.C. Vavalle J.P. Li L. J. Biol. Chem. 2000; 275: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Measurements of GH secretion were performed 48 h after transfection. PC12 cells were washed with a physiological salt solution (PSS (in mm): 145 NaCl, 5.6 KCl, 2.2 CaCl2, 0.5 MgCl2, 10 glucose, 15 HEPES, pH 7.4). The cells were then incubated for 15 min at 37 °C in PSS or PSS containing 56 mm KCl and 95 mm NaCl. The amounts of GH released into the medium and retained in the cells were determined by using a radioimmunoassay kit (Nichols Institute). To identify novel proteins that regulate SNARE function in neuronal cells, we used the full-length mouse SNAP-25b as bait to screen a two-hybrid rat hippocampal/cortical cDNA library. One of the positive clones was shown to encode part of a novel protein that we referred to as Spring because it is aSNAP-25-interacting RING finger protein. Re-transformation experiments confirmed that Spring interacts specifically with SNAP-25 but not with irrelevant baits such as synaptophysin nor with other neuronal SNAREs such as syntaxin 1 and VAMP2 (data not shown; see Fig. 4A). Moreover, our yeast two-hybrid interaction studies (not shown) and in vitrobinding data (Fig. 4A) reveal that, unlike several other known SNAP-25-interacting proteins such as syntaxins, SNIP, and intersectin, Spring does not interact with SNAP-23/syndet, a ubiquitously expressed SNAP-25 homologue (23Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar). Because the t-SNARE coiled-coil domains of SNAP-25 share significant homology with the t-SNARE domains in SNAP-23/syndet, syntaxin 1, and VAMP2 (24Weimbs T. Low S.H. Chapin S.J. Mostov K.E. Bucher P. Hofmann K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3046-3051Crossref PubMed Scopus (236) Google Scholar), the inability of Spring to interact with these other SNARE proteins further confirms the specificity of observed Spring-SNAP-25 interaction. By screening a λZAPII rat hippocampal cDNA library, we isolated three full-length and nine independent overlapping partial Spring cDNA clones. The full-length Spring cDNA contains an in-frame stop codon upstream of the initiator ATG codon with a Kozak consensus sequence and a single open reading frame encoding 710 amino acids in length, with a calculated molecular mass of 79.2 kDa (Fig. 1A). Spring is a hydrophilic protein with a theoretical isoelectric point (pI) of 6.54 and a high percentage (22%) of charged amino acids over the entire length. It contains neither a signal sequence nor a potential transmembrane domain. Analysis of Spring protein sequence reveals the presence of a RING finger domain followed by two B box motifs and a coiled-coil domain (Fig. 1), indicating that Spring is a new member of the RING-B box coiled-coil (RBCC) subfamily of RING finger proteins (25Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 26Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Crossref PubMed Scopus (357) Google Scholar). The RING finger and the B box motifs are cysteine/histidine-rich Zn2+-binding domains that are thought to mediate protein-protein interactions (27Borden K.L. Biochem. Cell Biol. 1998; 76: 351-358Crossref PubMed Scopus (234) Google Scholar). Although the function of the RBCC motif is unclear, this tripartite motif is found in a growing number of proteins involved in diverse cellular processes, from gene transcription and signal transduction to organelle transport (25Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 26Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Crossref PubMed Scopus (357) Google Scholar,28El-Husseini A.E. Vincent S.R. J. Biol. Chem. 1999; 274: 19771-19777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It has been proposed that the RBCC motifs may act as molecular building blocks in formation of large macromolecular scaffolds for these complex biological processes (26Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Crossref PubMed Scopus (357) Google Scholar). In addition to the RBCC motif, Spring contains a fibronectin type III domain, an autonomously folded protein module that is thought to mediate protein-protein interactions in both intracellular and extracellular compartments (29Spitzfaden C. Grant R.P. Mardon H.J. Campbell I.D. J. Mol. Biol. 1997; 265: 565-579Crossref PubMed Scopus (92) Google Scholar). At the C terminus, Spring has an SPRY domain, a putative protein-protein interaction module that was originally identified in thespla kinase and the ryanodine receptor (30Ponting C. Schultz J. Bork P. Trends Biochem. Sci. 1997; 22: 193-194Abstract Full Text PDF PubMed Scopus (172) Google Scholar). The SPRY domain has been found at the C terminus of several RBCC proteins with a conserved spacing between these two domains (31Seto M.H. Liu H.L. Zajchowski D.A. Whitlow M. Proteins. 1999; 35: 235-249Crossref PubMed Scopus (48) Google Scholar), although the significance of such a domain organization is not understood. Data base searches revealed the presence of Spring homologues as uncharacterized cDNAs or open reading frames obtained from genome projects in a number of organisms, including human, mouse,Drosophila, Caenorhabditis elegans, and zebrafish. The amino acid sequence of rat Spring is 98% identical to a recently published human sequence TRIM9, one of the 37 RBCCtripartite motif-containing proteins identified by dbEST data base searches with a consensus of the B box domain (32Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1053) Google Scholar). Moreover, the overall sequence of Spring protein shares 41 and 47% identity with C. elegans hypothetical protein C39F7.2 (GenBankTM accession number T33778 and AC006906) and a putative protein deduced from the genomic sequence ofDrosophila (a splice product of CG13145 and CG6256 genes, GenBankTM accession number AE003629), respectively (Fig. 1B). The conspicuous homology and conserved domain structure among Spring homologues from different species indicate that Spring is an evolutionarily conserved protein. Northern blot analysis of Spring mRNA expression revealed the presence of a major Spring transcript of 5.6 kilobase pairs and a minor form of 4.8 kilobase pairs, which may represent the products of alternative splicing or differential polyadenylation (Fig.2A). Spring mRNAs were prominently expressed in rat brain but were undetectable in the other tissues examined. Consistent with this result, analysis of human Spring mRNA expression using a Multiple Tissue Expression Array showed that Spring mRNA(s) was exclusively expressed in fetal and adult human brain where it was widely distributed in all brain regions tested (Fig. 2B). To characterize Spring protein, a rabbit anti-Spring antibody was generated against a 14-amino acid peptide of Spring. The anti-Spring antibody, but not preimmune serum, specifically recognized an 80-kDa protein in cells transfected with HA-tagged, full-length Spring cDNA, whereas no immunoreactivity was detected in vector-transfected control cells (Fig. 2C). The same 80-kDa band was also detected using the anti-HA antibody (data not shown). In rat brain, the anti-Spring antibody recognized a major 80-kDa band of endogenous Spring (Fig. 2, C and D). Occasionally, some minor bands of lower molecular weights were observed in brain homogenates as well as in cells expressing recombinant Spring protein (Fig. 2, C and D). These minor bands are likely to be the degradation products of the Spring protein because their relative intensity as compared with the 80-kDa band varied from preparation to preparation. Pre-absorption of the anti-Spring antibody with recombinant Spring protein completely eliminated its immunoreactivity to both recombinant and endogenous Spring protein (data not shown), confirming the specificity of the antibody. Western blot analysis of multiple rat tissues showed that Spring was expressed exclusively in brain (Fig. 2D), which is consistent with the pattern of Spring mRNA expression (Fig. 2A). To determine the intracellular distribution of Spring, rat brain postnuclear supernatant was separated into cytosol and membrane particulate fractions and then subjected to Western blot analysis with the anti-Spring antibody (Fig.3A). Although the primary structure of Spring does not contain any transmembrane domain, a large pool of Spring was found in the membrane fraction. The membrane-associated Spring could be extracted by 1.5 mNaCl, 8 m urea, or a pH 11.5 solution, suggesting that it is peripherally associated with membranes. Surprisingly, the membrane-associated Spring was resistant to solubilization by 4% Triton X-100 (Fig. 3A), suggesting that it may be associated with cytoskeleton. To examine this possibility, we used a well established protocol to isolate directly the cytoskeleton fractions from brain (18Stam J.C. Sander E.E. Michiels F. van Leeuwen F.N. Kain H.E. van der Kammen R.A. Collard J.G. J. Biol. Chem. 1997; 272: 28447-28454Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The integrity of these fractions was confirmed by immunoblotting with antibodies against actin, synaptophysin, and SNAP-25 (Fig. 3B). Immunoblot analysis of these fractions with the anti-Spring antibody revealed the presence of a substantial amount of Spring in the cytoskeleton fractions, indicating that a significant percentage of Spring is associated with brain cytoskeleton. To examine the subcellular distribution of Spring in more detail, synaptosome fractions were isolated and furt" @default.
• W2023981624 created "2016-06-24" @default.
• W2023981624 creator A5000323703 @default.
• W2023981624 creator A5006264996 @default.
• W2023981624 creator A5027222229 @default.
• W2023981624 creator A5074870071 @default.
• W2023981624 date "2001-11-01" @default.
• W2023981624 modified "2023-10-01" @default.
• W2023981624 title "Spring, a Novel RING Finger Protein That Regulates Synaptic Vesicle Exocytosis" @default.
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