Staring, a Novel E3 Ubiquitin-Protein Ligase That Targets Syntaxin 1 for Degradation

Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway. Syntaxin 1 is an essential component of the neurotransmitter release machinery, and regulation of syntaxin 1 expression levels is thought to contribute to the mechanism underlying learning and memory. However, the molecular events that control the degradation of syntaxin 1 remain undefined. Here we report the identification and characterization of a novel RING finger protein, Staring, that interacts with syntaxin 1. Staring is expressed throughout the brain, where it exists in both cytosolic and membrane-associated pools. Staring binds and recruits the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 and facilitates the ubiquitination and proteasome-dependent degradation of syntaxin 1. These findings suggest that Staring is a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation by the ubiquitin-proteasome pathway. Modulation of protein degradation is a major mechanism by which cells regulate the expression levels of specific proteins and consequently the cellular processes that these proteins participate in (1Schwartz A.L. Ciechanover A. Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (379) Google Scholar, 2Ciechanover A. Isr. Med. Assoc. J. 2001; 3: 319-327PubMed Google Scholar). The ubiquitin-proteasome pathway plays a crucial role in the degradation of proteins involved in a variety of cellular processes, including differentiation, proliferation, and apoptosis. However, the role of the ubiquitin-proteasome pathway in the degradation of presynaptic proteins remains poorly characterized, despite the presence of ubiquitin at nerve terminals (3Chapman A.P. Courtney S.C. Smith S.J. Rider C.C. Beesley P.W. Biochem. Soc. Trans. 1992; 20: 155SCrossref PubMed Scopus (14) Google Scholar, 4Chapman A.P. Smith S.J. Rider C.C. Beesley P.W. Neurosci. 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Ubiquitination involves a highly specific enzyme cascade in which ubiquitin is first activated by an E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; GST, glutathioneS-transferase; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; SNARE, soluble NSF attachment protein receptors. ubiquitin-activating enzyme and then transferred to an E2 ubiquitin-conjugating enzyme and finally ligated to the substrate by an E3 ubiquitin-protein ligase (1Schwartz A.L. Ciechanover A. Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (379) Google Scholar,7Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar, 8Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1200) Google Scholar). The E3 ubiquitin-protein ligase plays an essential role in determining the specificity of ubiquitination and subsequent protein degradation. Consistent with this role, it is estimated that an organism such as a human contains over 100 E3 ubiquitin ligases, in contrast to a single E1 ubiquitin-activating enzyme and about a dozen E2 ubiquitin-conjugating enzymes (9Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6958) Google Scholar). Despite the importance of E3 ubiquitin-protein ligases in specific protein degradation and the estimated presence of more than 100 E3 ligases in the human genome, only a few E3 ligases have been characterized at the molecular level. Syntaxin 1 is a neuronal membrane protein that was originally identified as a binding partner for synaptotagmin and the N-type calcium channel (10Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1077) Google Scholar, 11Inoue A. Akagawa K. Biochem. Biophys. Res. Commun. 1992; 187: 1144-1150Crossref PubMed Scopus (43) Google Scholar, 12Yoshida A. Oho C. Omori A. Kuwahara R. Ito T. Takahashi M. J. Biol. Chem. 1992; 267: 24925-24928Abstract Full Text PDF PubMed Google Scholar). It is well established that syntaxin 1 functions as a synaptic t-SNARE to mediate synaptic vesicle exocytosis at nerve terminals (13Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar, 14Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1025) Google Scholar, 15Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (422) Google Scholar). Syntaxin 1 appears early during embryonic development (16Kushima Y. Fujiwara T. Sanada M. Akagawa K. J. Mol. Neurosci. 1997; 8: 19-27Crossref PubMed Scopus (24) Google Scholar, 17Noakes P.G. Chin D. Kim S.S. Liang S. Phillips W.D. J. Comp. Neurol. 1999; 410: 531-540Crossref PubMed Scopus (29) Google Scholar), and its expression level is dramatically up-regulated during synapse formation and brain maturation (16Kushima Y. Fujiwara T. Sanada M. Akagawa K. J. Mol. Neurosci. 1997; 8: 19-27Crossref PubMed Scopus (24) Google Scholar, 17Noakes P.G. Chin D. Kim S.S. Liang S. Phillips W.D. J. Comp. Neurol. 1999; 410: 531-540Crossref PubMed Scopus (29) Google Scholar, 18Miya F. Yamamoto A. Akagawa K. Kawamoto K. Tashiro Y. Cell Struct. Funct. 1996; 21: 525-532Crossref PubMed Scopus (7) Google Scholar, 19Veeranna Grant P. Pant H.C. Dev. Neurosci. 1997; 19: 172-183Crossref PubMed Scopus (22) Google Scholar). Regulation of syntaxin 1 levels may contribute to the mechanism underlying learning and memory, since changes in syntaxin 1 levels have been found to correlate with long term potentiation and various learning and memory behaviors (20Davis S. Rodger J. Hicks A. Mallet J. Laroche S. Eur. J. Neurosci. 1996; 8: 2068-2074Crossref PubMed Scopus (29) Google Scholar, 21Hicks A. Davis S. Rodger J. Helme-Guizon A. Laroche S. Mallet J. Neuroscience. 1997; 79: 329-340Crossref PubMed Scopus (63) Google Scholar, 22Richter-Levin G. Thomas K.L. Hunt S.P. Bliss T.V. Neurosci. Lett. 1998; 251: 41-44Crossref PubMed Scopus (26) Google Scholar). Alteration in syntaxin 1 expression levels has been associated with several neurodegenerative diseases and psychiatric disorders, including schizophrenia, Alzheimer's disease, and Creutzfeldt-Jakob disease (23Gabriel S.M. Haroutunian V. Powchik P. Honer W.G. Davidson M. Davies P. Davis K.L. Arch. Gen. Psychiatry. 1997; 54: 559-566Crossref PubMed Scopus (125) Google Scholar, 24Shimohama S. Kamiya S. Taniguchi T. Akagawa K. Kimura J. Biochem. Biophys. Res. Commun. 1997; 236: 239-242Crossref PubMed Scopus (83) Google Scholar, 25Honer W.G. Falkai P. Young C. Wang T. Xie J. Bonner J., Hu, L. Boulianne G.L. Luo Z. Trimble W.S. Neuroscience. 1997; 78: 99-110Crossref PubMed Scopus (140) Google Scholar, 26Ferrer I. Rivera R. Blanco R. Marti E. Neurobiol. Dis. 1999; 6: 92-100Crossref PubMed Scopus (45) Google Scholar). Despite the importance of the regulation of syntaxin 1 levels in synaptic function and dysfunction, the molecular mechanisms underlying such regulation remain undefined. To identify proteins that regulate syntaxin 1, we carried out a search in rat brain for proteins that interact with syntaxin 1 using yeast two-hybrid screens. Here we report the isolation of a novel syntaxin 1-interacting protein, named Staring, that acts as an E3 ubiquitin-protein ligase to promote the ubiquitination and degradation of syntaxin 1 by the proteasome pathway. The bait plasmid, pPC97-Syntaxin 1, was constructed by subcloning the cytoplasmic domain (amino acids 5–270) of rat syntaxin 1B (27Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W., Li, Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar) into the pPC97 vector (28Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (481) Google Scholar, 29Li X.J., Li, S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar). For the two-hybrid screen, the yeast strain CG-1945 (CLONTECH) was transformed sequentially with pPC97-Syntaxin 1 and a rat hippocampal/cortical two-hybrid cDNA library (29Li X.J., Li, S.H. Sharp A.H. Nucifora Jr., F.C. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (541) Google Scholar), using the lithium acetate method (30Chin 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). Positive clones were selected on 3-aminotriazole (5 mm; Sigma)-containing medium lacking leucine, tryptophan, and histidine and verified with a filter assay for β-galactosidase activity. Prey plasmids were then recovered and retransformed into yeast with pPC97-Syntaxin 1 or various control baits to confirm the specificity of the interaction. For cloning of full-length Staring, a partial Staring cDNA probe from the prey clone (clone 7) was used to screen a rat hippocampal cDNA library in λZAPII (Stratagene) according to the standard procedure (31Sambrook 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 Staring clones were sequenced multiple times on both strands using an Applied Biosystems 373A DNA sequencer. Conventional molecular biological techniques (31Sambrook 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 subclone DNA fragments encoding full-length and truncated forms of Staring into the following vectors: the pPC97 and pPC86 vectors for yeast two-hybrid interaction studies; the prokaryotic expression vectors pGEX-5X-2 (Amersham Biosciences) and pET28c (Novagen) for the production of GST- and His6-tagged fusion proteins; and the mammalian expression vectors pCDNA3.1(+) (Invitrogen) and pCHA (30Chin 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 HeLa cells. The expression construct pRK5-HA-UbcH5, pRK5-HA-UbcH7, and pRK5-HA-UbcH8 were obtained as generous gifts from Dr. Ted Dawson (32Zhang Y. Gao J. Chung K.K. Huang H. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13359Crossref PubMed Scopus (842) Google Scholar). Four polyclonal anti-Staring antibodies, two in chicken (CS-N and CS-C) and two in rabbit (RS-N and RS-C), were generated against Staring N-terminal peptide MSGLSNKRAAGDGG and C-terminal peptide AAFGAHDFHRVYIS, respectively. The antibodies were affinity-purified using the immunogen peptide-coupled columns as described previously (30Chin 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). Other antibodies used in this study include the following: anti-HA (3F10 (Roche Molecular Biochemicals) and HA.11 (Covance)), anti-Myc (9E10.3; Neomarkers), anti-syntaxin 1 (HPC-1; Sigma), anti-actin (C4; Roche Molecular Biochemicals) and secondary antibodies coupled with horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc.). Northern blot analysis of Staring 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 Staring cDNA fragment from clone 7 as the probe (31Sambrook 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-Staring and other antibodies. Antibody binding was detected by using the enhanced chemiluminescence system (Amersham Biosciences). Subcellular fractionations of rat brain into cytosol fraction (100,000 × gsupernatant) and membrane fraction (100,000 × gpellet) were preformed as previously described (30Chin 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 studies as described (30Chin 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), using 1.5 m NaCl or 4 m urea. GST-Staring fusion proteins or GST control were immobilized on glutathione-agarose beads (Sigma) and incubated with rat brain homogenates as previously described (30Chin 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, 33Li Y. Chin L.S. Weigel C. Li L. J. Biol. Chem. 2001; 276: 40824-40833Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). After extensive washes, bound proteins were eluted by boiling in 2× Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting with appropriate antibodies. HeLa or SH-SY5Y cells were transfected with indicated plasmids using LipofectAMINE (Invitrogen) as described by the manufacturer. Cell lysates were prepared and subjected to immunoprecipitation as described previously (34Chin 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 (166) Google Scholar), using anti-HA antibody (3F10), anti-Myc (9E10.3), anti-Staring (RS-N), anti-syntaxin 1 antibody (HPC-1), or control IgG. The immunocomplexes were recovered by incubation with protein G- or protein A-Sepharose beads (Sigma). After extensive washes, the immunocomplexes were dissociated by boiling in the Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting. HeLa cells were transfected with combinations of the following plasmids: pCHA-syntaxin 1, pcDNA3-Myc-ubiquitin, pFLAG-Staring, and pFLAG-StaringΔR, a C-terminal deletion mutant of Staring that lacks the RING finger motif. SH-SY5Y cells were transfected with pcDNA3-Myc-ubiquitin in combination with pFLAG-Staring or pFLAG-StaringΔR. Twenty-four hours after transfection, the cells were incubated for 8 h with 20 μm MG132 (Calbiochem). The cells were then lysed, and an equal amount of protein from each lysate was immunoprecipitated using antibodies against HA tag or syntaxin 1 (34Chin 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 (166) Google Scholar). Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with an anti-Myc antibody to detect Myc-ubiquitin conjugated to syntaxin 1. HeLa cells were co-transfected with pCHA-syntaxin 1 and either pFLAG-Staring or pFLAG vector control. SH-SY5Y cells were transfected with pFLAG-Staring or pFLAG vector control. To control for the transfection efficiency, cells were transfected using LipofectAMINE in a 150-mm culture dish. At 24 h post-transfection, the cells were divided into eight 60-mm dishes, each of which was used for a single chase time point. At 48 h post-transfection, cells in the 60-mm dishes were washed and incubated for 30 min with Met/Cys-free DMEM. The medium was then replaced with Met/Cys-free DMEM containing 200 μCi of [35S]Met/Cys (1000 Ci/mmol) express protein labeling mix (PerkinElmer Life Sciences). After incubation for 1 h, the radioactive medium was removed by extensive washes with nonradioactive DMEM. Cells in each 60-mm dish were then incubated for the indicated chase time in nonradioactive DMEM supplemented with 10% fetal bovine serum and 5 times the normal concentration of methionine and cysteine as described previously (35Wheeler T.C. Chin L.S., Li, Y. Roudabush F.L. Li L. J. Biol. Chem. 2002; 277: 10273-10282Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Cells were then lysed, and an equal amount of protein from each lysate was immunoprecipitated using an anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE and analyzed by a PhosphorImager (Amersham Biosciences). HeLa or SH-SY5Y cells expressing Staring and syntaxin 1 were incubated for 8 h at 37 °C with the proteasome inhibitor MG132 (20 μm;Calbiochem), the cysteine protease inhibitor E-64 (50 μm; Sigma), the lysosomal protease inhibitor NH4Cl (50 mm) or chloroquine (100 μm; Sigma), or vehicle (Me2SO; final concentration 0.1%). Cells were then lysed, and the protein concentrations of the lysates were determined by the BCA protein assay (Pierce). An equal amount of protein from each lysate was then analyzed by SDS-PAGE and immunoblotting. To identify syntaxin 1-interacting proteins, we screened a rat hippocampal/cortical cDNA library by yeast two-hybrid selection using the cytoplasmic domain of rat syntaxin 1B as bait. This screen led to the isolation of several clones encoding SNAP-25, a known syntaxin 1-interacting protein (data not shown), confirming the validity of the two-hybrid screen. One of the positive clones was shown to encode part of a novel protein that we referred to as Staring (because it is asyntaxin 1-interacting RING finger protein (Fig. 1 A). Retransformation experiments confirmed that Staring interacts specifically with syntaxin 1A and syntaxin 1B but not with SNAP-25 or SNAP-23 (data not shown). Since SNAP-25 and SNAP-23 contain coiled-coil t-SNARE domains that are homologous to the t-SNARE domain of syntaxin 1 (36Weimbs 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 Staring to interact with SNAP-25 and SNAP-23 further confirms the specificity of the Staring-syntaxin 1 interaction. Cloning of the full-length Staring cDNA revealed that Staring is a 1002-amino acid protein with a calculated molecular mass of 113.8 kDa. The sequence surrounding the initiator methionine codon of Staring conforms well to the translation initiation consensus sequence (37Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4172) Google Scholar) and is preceded by an in-frame stop codon in the 5′-untranslated region. Furthermore, the coding sequence of Staring beginning with this methionine initiator can be expressed in mammalian cells to yield a recombinant protein with an apparent molecular weight similar to that of endogenous Staring (data not shown), confirming that the cloned Staring sequence contains the entire coding region. Staring is highly hydrophilic, with a theoretical isoelectric point (pI) of 6.13 and a high percentage (34%) of charged amino acids over the entire length. Staring contains neither a signal sequence nor a potential transmembrane domain. As shown in Fig. 1, Staring contains six putative coiled-coil domains and a RING finger motif at the C terminus. The RING finger motif is a cysteine/histidine-rich (C3HC4), Zn2+ binding domain that is found in a number of eukaryotic proteins, some of which have been implicated in vesicular transport (38Saurin 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). Emerging evidence indicates that the RING finger motif may function in protein ubiquitination as a key determinant of the E3 ubiquitin-protein ligase activity (39Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Crossref PubMed Scopus (357) Google Scholar, 40Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar, 41Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). The N-terminal region of rat Staring is 90% identical to human RBP95, an 838-amino acid protein recently identified from a yeast two-hybrid screen using retinoblastoma protein as bait (42Wen H. Ao S. Biochem. Biophys. Res. Commun. 2000; 275: 141-148Crossref PubMed Scopus (5) Google Scholar). The expression of endogenous RBP95 protein has not yet been characterized; however, based on EST data base searches, RBP95 seems to be a rare isoform derived from alternative splicing of the gene encoding the uncharacterized human ring finger protein 40 (RNF40, also called KIAA0661) (Fig.2 A). Data base searches reveal the presence of Staring homologues as uncharacterized cDNAs or open reading frames obtained from genome projects in a number of organisms, including humans, mice,Drosophila, Caenorhabditis elegans,Arabidopsis, and yeast (Fig. 2 A). Sequence comparison analysis indicates that RNF40 is the human orthologue of rat Staring. The human genome also contains a second Staring homologue, RNF20, which is encoded by a gene that is distinct from the RNF40 gene. In Drosophila, C. elegans,Arabidopsis, and yeast, there appears to be only one Staring homologue. Whereas the function of these Staring homologues is unknown, the deletion mutant of the yeast Staring homologue, Bre1p (also called YDL074c), was reported to be sensitive to multiple drugs, including brefeldin A and chlorpromazine (43Rieger K.J., El- Alama M. Stein G. Bradshaw C. Slonimski P.P. Maundrell K. Yeast. 1999; 15: 973-986Crossref PubMed Google Scholar, 44Muren E. Oyen M. Barmark G. Ronne H. Yeast. 2001; 18: 163-172Crossref PubMed Scopus (49) Google Scholar). Analysis of the deduced proteins from the Staring-homologous sequences reveals that these homologous proteins have a domain structure that is similar to that of Staring (Fig. 2 A). Most notably, these Staring homologues contain a highly conserved RING finger motif at their C terminus (Fig.2 B). The conspicuous homology and conserved domain structure among Staring homologues from different species indicate that Staring is an evolutionarily conserved protein. Northern blot analysis of Staring mRNA expression revealed the presence of a single Staring transcript of 5.1 kb (Fig.3 A). The Staring mRNA is relatively abundant in brain, testis, heart, liver, and kidney and expressed at low levels in lung, spleen, and skeletal muscle. Consistent with this result, analysis of human Staring mRNA expression using a multiple tissue expression array showed that Staring mRNA is ubiquitously expressed in various brain regions as well as all fetal and adult human tissues examined (data not shown). The broad tissue distribution of Staring mRNA expression suggests that Staring has a functional role important to many cell types, including neurons. To characterize Staring at the protein level, we generated four polyclonal anti-Staring antibodies, two in chicken (CS-N and CS-C) and two in rabbit (RS-N and RS-C), against the N- and C-terminal 14-amino acid peptide of rat Staring, respectively. The antibodies (CS-N and RS-N) against the N terminus of Staring are expected to detect both Staring and RBP95 isoforms, whereas the antibodies (CS-C and RS-C) against the C terminus of Staring should only recognize the Staring isoform. Western blot analysis demonstrated that all four anti-Staring antibodies, but not their corresponding preimmune controls, recognized a single endogenous protein band of ∼125 kDa (Fig. 3, Band C; data not shown), indicating that Staring is the predominant isoform expressed in rat. No 95-kDa protein band corresponding to RBP95 could be detected by either chicken (CS-N) or rabbit (RS-N) antibodies against the N terminus of Staring (Fig.3 B; data not shown), suggesting that RBP95 is a rare isoform that is either expressed at extremely low levels or not expressed at all. All four anti-Staring antibodies specifically react with recombinant Staring protein expressed in bacterial and mammalian cells (data not shown). Furthermore, preabsorption of these anti-Staring antibodies with recombinant Staring protein completely eliminated their immunoreactivity to recombinant as well as endogenous Staring protein (data not shown), confirming the specificity of these antibodies. In agreement with the result of Northern blot analysis (Fig. 3 A), Western blot analysis using the anti-Staring antibodies revealed that Staring protein is ubiquitously expressed in all tissues tested, although the expression levels in heart and skeletal muscle are very low (Fig. 3 B). To examine the intracellular distribution of endogenous Staring, postnuclear supernatant of rat brain was separated into cytosol and membrane fractions and then subjected to Western blot analysis with anti-Staring antibodies (Fig. 3 C). Staring immunoreactivity was detected in both cytosol and membrane fraction, although the relative amount of Staring in the cytosol fraction was severalfold more than that in the membrane fraction. To investigate the nature of Staring association with membranes, the membrane fraction was extracted with 1.5 m NaCl or 4 m urea (Fig.3 C). Unlike the integral membrane protein syntaxin 1 that was resistant to extraction by high salt and urea, a majority of Staring was extracted by these treatments, suggesting that Staring is peripherally associated with membranes via hydrophilic interactions. To determine whether the Staring-syntaxin 1 interaction detected in yeast actually takes place in vitro, GST fusion proteins containing various portions of Staring (Fig.4 A) were immobilized on glutathione beads and used to bind endogenous syntaxin 1 from rat brain homogenate. As shown in Fig. 4 B, the GST-fusion proteins bearing the full-length Staring or the Staring fragments that contain the predicted coiled-coil domain H3 (Staring Δ1 and Staring Δ2) was able to bind endogenous syntaxin 1. In contrast, the GST-Staring fusion protein containing the coiled-coil domain H4 (Staring Δ3) or GST alone was unable to pull down syntaxin 1, confirming that the observed Staring-syntaxin 1 interaction is specific. These data indicate that the syntaxin 1-binding site of Staring lies within the H3 domain, between amino acid residues 448 and 536. In addition, other parts of Staring seem to also contribute to the interaction with syntaxin 1, since the GST-fusion proteins containing truncated forms of Staring bound much less syntaxin 1 than a similar amount of the full-length GST-Staring (Fig. 4 A). To determine whether Staring associates with syntaxin 1 in vivo, we first performed co-immunoprecipitation experiments using lysates of HeLa cells expressing exogenous syntaxin 1 and HA-tagged Staring. As shown in Fig. 4 C, syntaxin 1 and HA-Staring were co-immunoprecipitated by the anti-HA antibody, providing evidence for the association of these two proteins in mammalian cells. By comparison, control IgG was unable to precipitate either syntaxin 1 or Staring. We then performed additional co-immunoprecipitation experiments to examine the association of endogenous Staring and syntaxin 1 in rat brain synaptosomes (Fig. 4 D). Anti-syntaxin 1 antibody, but not the mouse IgG control, was able to co-immunoprecipitate syntaxin 1 and Staring from solubilized synaptosomes, demonstrating the existence of endogenous Staring-syntaxin 1 complexes at nerve terminals. Under our experiment conditions, ∼10% of total endogenous Staring was co-immunoprecipitated with syntaxin 1, indicating that only a fraction of Staring and syntaxin 1 co-exist in the Staring-syntaxin 1 complexes. These results are consistent with previous reports that syntaxin 1 interacts with more than a dozen proteins, including SNAP-25, nSec1/Munc-18, Munc-13, tomosyn, and syntaphilin (14Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1025) Google Scholar, 45Hata Y. Slaughter C.A. Sudhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (594) Google Scholar, 46Betz A. Okamoto M. Benseler F. Brose N. J. Biol. Chem. 1997; 272: 2520-2526Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 47Fujita Y. 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