with syntaxin

Proc. Nad. Acad. Sci. USA Vol. 91, pp. 2003-2007, March 1994 Cell Biology

A rat brain Secl homologue related to Rop and UNC18 interacts with syntaxin (synaptic vesicle/exocytosis/VAMP/synaptobrevin/SNAP-25)

ELIZABETH P. GARCIA, EVELINA GATTI, MARGARET BUTLER, JANET BURTON, AND PIETRO DE CAMILLI* Department of Cell Biology and Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510

Communicated by Vincent Marchesi, December 7, 1993 (received for review November 19, 1993)

factors required for fusion including N-ethylmaleimidesensitive fusion proteins, a-, /-, and y-SNAPs (3). Corresponding yeast homologues, which play a general role in membrane fusion in the secretory pathway, have also been identified (15-17). It is quite clear, however, that additional components must be involved in the fusion machinery. For example, work in yeast has defined several gene products that participate in exocytosis and for which there are no identified mammalian homologues (18). One such protein is Secl (19, 20). Secl has sequence similarity to two other yeast proteins, Slpl (VPS33) and Slyl, which are thought to operate in vesicular traffic from the Golgi complex to the vacuole (21, 22) and from the endoplasmic reticulum to the Golgi complex (23, 24), respectively. Two proteins that share sequence similarity with Secl have been recently identified in Drosophila melanogaster and in Caenorhabditis elegans. The Drosophila Secl homologue, called Rop (Ras opposite), was identified as the protein encoded by a gene that is localized next to the RAS2 gene and is transcribed in the opposite direction but under the control of the same promoter. Rop is expressed in the nervous system and in selected tissues with high secretory and endocytic activity (25). The C. elegans Secl homologue, UNC18, is a protein whose mutations lead to a phenotype consistent with an impairment of presynaptic function. UNC18 was found to be expressed selectively in the nervous system (26-28). It appears, therefore, that Secl-related proteins may define a family of proteins that play an essential role in membrane docking and/or fusion, including the exocytosis of synaptic vesicles. Elucidation of the function of these proteins awaits the study of their biochemical interactions. The recent demonstration that SS01 and SS02, two yeast genes encoding syntaxin homologues, are high copy suppressors of mutations in the SEC] gene (10) suggests the attractive possibility that proteins of the syntaxin family interact directly with proteins of the Secl family. The goal of this study was to identify a rat brain homologue of Secl and to determine whether this protein interacts with syntaxin.t

ABSTRACT Secl is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Secl gene, SSOI and SS02, were recently identified that encode proteins of the syntaxin family. Syntain (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Secl were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabdiis ekgans (UNC18). Based on the amino acid sequence alignment of Secl, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Secl homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of593 amino acids (rbSecl). Antibodies raised against a central portion of rbSecl recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSecl-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specificaly interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Secl family in membrane fusion involves an interaction with a T-SNARE.

Elucidation of the molecular mechanisms by which synaptic vesicles dock and fuse with the plasmalemma has recently been the focus of intense investigation (1). Increasing evidence suggests that molecular mechanisms of synaptic vesicle exocytosis are fundamentally similar to mechanisms that operate in all types of exocytosis and, more generally, in all membrane fusion events in the secretory and endocytic pathway (2). A set of components that appear to form the core of the docking and fusion machinery for synaptic vesicles has been identified. According to a recently proposed model, referred to as the SNARE hypothesis (3), the synaptic vesicle protein synaptobrevin/VAMP (referred to as a V-SNARE) (4, 5) interacts with syntaxin (6) and SNAP-25 (referred to as T-SNARES) (7), two proteins localized in the plasmalemma (3, 8). (VAMP is a vesicle-associated membrane protein; SNAP is a soluble N-ethylmaleimide-sensitive attachment protein fusion protein; and SNARE is a SNAP receptor.) Homologues of each of these proteins are present in yeast, and genetic analysis has demonstrated their participation in exocytosis (refs. 9-11; V. Bankaitis, personal communication). Furthermore, the crucial role of these proteins in neuronal exocytosis is emphasized by the demonstration that they are targets for the proteolytic action of clostridial neurotoxins, which are potent inhibitors of neurotransmitter release (12-14). Formation of the SNARE complex is then thought to be followed by the recruitment of several cytosolic

MATERIALS AND METHODS Cloning and Sequencing of rbSecl cDNA. Two degenerate oligonucleotide primers (AHVFFT and DAEGE, corresponding to amino acids 113-118 and 386-390 of Rop, respectively) were synthesized based on the protein sequence alignment of Rop, UNC18, and Secl (W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University). PCRs were carried out using 0.2 gg of a rat brain cDNA Abbreviations: rbSecl, rat brain Secl; GST, glutathione S-transferase. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U06069).

The publication costs of this article were defrayed in part by page charge

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2003

2004

Cell Biology: Garcia et al.

library (29). A DNA product of the expected size was amplified (860 bp). This product was subcloned into pBluescript II SK- (Stratagene), sequenced by standard methods (30, 31), and found to encode a sequence related to SEC]. The PCR fragment was radiolabeled with [yt32P]dCTP (Amersham) by random priming (Boehringer Mannheim) and used to screen an oligo(dT)-primed A ZAP II rat brain cDNA library (Stratagene, no. 936501). Twenty-five nitroceliulose filters containing 20-30 x 103 phage plaques were screened (31). Fourteen putative positive clones were isolated and three plaques remained positive through sequential plaque purifications. Clones were subcloned into pBluescript II SK- plasmid (Stratagene) and sequenced in both directions by standard methods (30, 31). Northern Blot Analyts. Total RNA was isolated from tissues by the guanidine isothiocyanate procedure (32). Poly(A)+ RNA was isolated by oligo(dT)-cellulose and run on formaldehyde agarose gels (32). The RNA was transferred to Zeta-Probe nylon membrane (Bio-Rad) for 2 hr at 0.8 A in TAE (40 mM Tris acetate/2 mM EDTA, pH 8.0). The blot was hybridized for 16'hr at 3rC with [y-32P]dCTP randomprimed clone 16 cDNA in 50% formamide/5 x SSC (150 mM NaCl/15 mM trisodium citrate, pH 7.0)/160 mM potassium phosphate, pH 7.4/1 x Denhardt's reagent/50 pug of salmon sperm DNA per ml. High stringency washes were performed at 680C in 0.2x SSC/O. 1% SDS. Atibodies. A rabbit anti-rbSecl antiserum was obtained by injecting a recombinant protein composed of maltose binding protein (New England Biolabs) fused to amino acids 102-255 of rbSecl (see below). A rabbit anti-SNAP-25 antiserum was raised by using the protocol described by Qyler et al. (7). The following anibdies were generous gifts: a mouse monoclonal antibody directed against syntaxin from C. Barnstable (Yale University) (33), a rabbit anti-rab3A antiserum (34), and a monoclonal antibody directed against synaptobrevin (clone 10.3) (4) from R. Jahn (Yale University). Produton of rbSecl Fuion Proteins. Clone 16 was subcloned into the bacterial expression vector pGEX-2T (Pharmacia) obtaining an-in-frame recombinant protein composed of glutathione S-transferase (GST) fused to the N terminus of rbSecl. rbSecl-GST fusion protein and GST were produced in Escherichia coli, and soluble bacterial extracts containing rbSecl-GST fusion protein or GST protein were then affinity-purified on glutathione-Sepharose 4B beads (Pharmacia) (32). Another fusion protein composed of the maltose bindi protein fused to amino acids 102255 of rbSecl was produced in the pMAL-p expression vector (New England Biolabs). The cDNA encoding the rbSecl portion of the fusion protein was obtained by PCR using synthetic oligonucleotide primers flanked by BamHI and Xba I cleavage sites. RbSecl protein present in soluble bacterial extracts was affinity-purified on amylose resin (New England Biolabs) and used to immunize rabbits. Binding of rb&ecl-GST to Triton X-1O-WSolubillzed Rat Brain Exrac. Frozen rat brains (Pel-Freez Biologicals) were homogenized (1:20, wt/vol) in 150 mM NaCl/10 mM Hepes-KOH, pH 7.4, containing pepstatin, aprotinin, leupeptin, antipain (each at 4 pg/ml), 0.4 ;M phenylmethylsulfonyl fluoride, and 10 mM benzamidene using an SDT Tissumjzer (Tekmar, Cincinnati). Triton X-100 (Sigma) was then added, to a final concentration of 0.5% (vol/vol); the mixture was incubated at 40C for 1 hr with constant agitation and subsequently clarffied by centrifugation at 200,000 x g for'2 hr at 40C. Glutathione-Sepharose 4B beads preincubated with similar amounts of RbSecl-GST or GST were added to 1 ml of the supernatant and incubated with gentle mixing for 2 hr at 40C. Beads were then washed three times in ice-cold 150 mM NaCl/10 mM sodium phosphate, pH 7.4, with gentle mixing for 10 min and eluted with 10 mM Tris HCl, pH 7.4/5 mM glutathione (Sigma) for 30 min with gentle mixing.

Proc. Nati. Acad. Sci. USA 91 (1994) Eluates were separated from the beads by centrifligation at 13,000 x g and processed for SDS/PAGE and Western

blotting.

mProcedues. Tissues were homogenized and Nu_1ena processed for SDS/PAGE as described (35). SDS/PAGE and Western blotting were performed essentially as described by Laeinmli (36) and Towbin et al. (37). Antibodies coupled to alkaline phosphatase were used as secondary reagents for Western blotting.

RESULTS Degenerate PCR primers corresponding to amino acid sequences conserved among Secl, UNC18, and Rop were used to amplify a portion of a rat brain cDNA (860 bp) that encoded a sequence related to Secl (see Materials and Methods). This fragment was used to screen a A ZAP II rat brain cDNA library (Stratagene). Three positive clones were isolated, subcloned into pBluescript II SK-, and found to be 2.3 (clone 16), 1.8 (clone 24), and 3.0 (clone 22) kb long. Clones 16 and 24 were sequenced in their entirety. The deduced amino acid sequence of clone 16 is shown in Fig. 1. It encodes a methionine at nucleotide position 18. The co ding codon is embedded in the sequence AACGCCATG that fits the consensus defined by Kozak for an initiation codon (38). Eight of 10 nucleotides fit the consensus and. the two positions most critical for function (positions 4 and 10) are present. This methionine is followed by a sgle 1781-lbp open reading frame that terminates at position 1797 encdig a putative protein of 593 amino acids with a prdicted. Mr of 67,450, which we refer to as rbSecl. Clone 24 starts at position 23 of clone 16 and is identical to it until position 1718- i.e., 81 bases upstream of its stop codon. Beyond this position, clone 24 continues for an additional 45 bases, which have no homology to the cortesponding region of clone 16. Given the evidence for a single mRNA species (see below), this divergent region ofdione 24 may represent a cloning artifact although the possiiity that it may represent an alternatively spliced form cannot be excluded at this time. Restriction analysis and partial sequence of clone 22, which start at position 330 of clone 16, confirhed the sequence of clone 16. The amino acid sequence of the rbSecl exhibits a substantial similarity along its entire length to other proteins of the Seci family (Fig. 1). As shown in Table 1, rbSecl is most similar to Rop (25) (65%) and increasingly less similar to UNC18 (27) (59o), Secl (39) (27%), Slyl (22) (22%), and SIp1 (21) (20%). This hierarchy of similarity agrees with evolutionary considerations and is consistent with a role of rbSecl in-exocytosis. The overall size of all the proteins of the family is very similar and only Secl has an additional C-terminal extension. The N terminus of rbSecl aligns well with the N terminus of Rop UNC18 and Secl. This finding, together with the presence of a strong Kozak consensus (see above), supports the correct assignment of the start methionine in clone 16, the clone with the longest 5' extension, in spite of the lack of any stop codon in the short nucleotide sequence preceding this methionine. Salzberg et al. (25) identified a a-Cop motif in a 27-amino acid stretch of Rop, Secl, Slyl, and Slpl. This motif is also present in rbSecl and in UNC18. Like other members of the family, rbSecl is predicted to be a hydrophilic protein (pI 7.01), with a high content (311%) of charged amino acids and no putative transmembrane regions. The Swiss-Prot and the GenBank/EMBL data bases were searched for additional related sequences at the protein and nucleotide levels, respectively. The only substantial homology was found between the 3' end of clone 16 and the nucleotide sequence of a human' brain cPNA (GenBank. accession no. M79125) (40). The sequence of this partial cDNA contains at its 5' end an

Proc. Natl. Acad. Sci. USA 91 (1994)

Cell Biology: Garcia et al.

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FIG. 1. Sequence alignment ofrbSecl with other Secl-related proteins. Amino acid sequences are shown in single letter code. Identical amino acids at a given position are boxed. rbSecl sequence has been aligned with those of Rop (25), UNC18 (27), and Secl (20). Sequences were aligned by using the program PILEUP from the Genetics Computer Group. Amino acids shared between rbSecl and at least one other protein are boxed.

open reading frame encoding an amino acid sequence identical to the last 24 amino acids of rbSecl. These 24 amino acids are followed by a termination codon in both sequences. This observation strengthens the conclusion that the C terminus assigned to rbSecl by the sequence of clone 16 is correct. Northern blots of rat poly(A)+ mRNA from brain, heart, kidney, liver, and lung were probed with [y-32P]dCTP-labeled full-length clone 16 to determine the size and tissue distribution of the rbSecl mRNA. Under high stringency, we detected a single message of 3.5 kb selectively expressed in brain tissue (Fig. 2), indicative of a single rbSecl-related mRNA. Only after prolonged exposure of the autoradiogram, extremely faint bands with the same electrophoretic mobility Table 1. Sequence similarities among Secl-related rbSecl Uncl8 Secl Rop 0.27 rbSecl 0.65 0.59 0.27 0.65 0.59 Rop 0.26 Unc18 0.59 0.59 0.27 0.27 0.26 Secl 0.24 0.22 0.22 0.23 Slyl SIpi 0.20 0.20 0.21 0.20

proteins Sipi Slyl 0.22 0.22 0.23 0.24

0.19

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were observed in other tissues (data not shown). An antiserum raised against rbSecl (but not the preimmune serum) recognized a protein band with an apparent molecular mass

z


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FIG. 2. Northern blot analysis of rat tissues. Three micrograms of poly(A)+ mRNA from the indicated tissues or rRNA (isolated from lung) was applied to each lane. The blot was probed with randomprimed [-y-32P]dCTP-labeled clone 16 cDNA and washed under high stringency conditions as described.

Cell

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Proc. Natl. Acad. Sci. USA 91 (1994)

Biology: Garcia et al.

of 67 kDa in a brain extract probed by Western blotting (Fig. 3A). This size is in very good agreement with the predicted molecular mass of the protein (see above). This band was not detected in extracts of pituitary, pancreas, testis, heart, lung, kidney, liver, PC12 cells (41), and rat insulinoma cells (RINm5) (42) (Fig. 3B). We next investigated whether recombinant rbSecl could interact with syntaxin. A GST fusion protein that contains the entire open reading frame of rbSecl was constructed in pGEX2T vector. The apparent molecular mass of the protein in SDS/PAGE was 95 kDa as predicted by the coding sequence. Furthermore, rbSecl-GST protein was highly immunoreactive with anti-rbSecl antibody (data not shown). rbSecl-GST and GST immobilized on glutathione-Sepharose 4B beads were incubated with Triton X-100-solubilized brain extract. Proteins specifically bound to the beads were then eluted with glutathione and analyzed by SDS/PAGE and Western blotting using antibodies directed against V- and T-SNAREs implicated in synaptic vesicle exocytosis. As shown by Fig. 4, syntaxin, but not synaptobrevin/VAMP or SNAP-25, was specifically recovered on the rbSecl-GST beads and not by the GST beads. Genetic analysis of sec mutant yeast strains suggests that the SEC] and SLY] genes may interact with the SEC4 and YPTI genes, respectively (24, 43). Because YPTI and SEC4 are members of the rab family (44), we probed the material eluted from rbSecl-GST beads with antibodies directed against rab3A, a rab protein concentrated on synaptic vesicles (34). No rab3A was detected.

DISCUSSION Here we describe the identification of a rat brain protein, rbSecl, similar to the yeast Secl protein and demonstrate that rbSecl interacts selectively with syntaxin, one of the SNAREs involved in neurosecretion. This finding provides additional information regarding the molecular mechanisms involved in synaptic vesicle exocytosis. In addition, this result is likely to have implications for the elucidation of mechanisms of vesicle docking and fusion in other types of exocytosis and in vesicular traffic at other stations of the secretory pathway. Seci is a protein that participates selectively in exocytosis from yeast (19), while two related yeast proteins, Slyl (22) and Slpl (21), are involved in transport from the endoplasmic C.

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FIG. 4. Biochemical interaction of rbSecl with syntaxin. Equal amounts of rbSecl-GST or GST coupled to glutathione-Sepharose 4B beads were incubated with 10 mg of a Triton X-100-solubilized rat brain extract. Specifically bound proteins were eluted in 5 mM glutathione/10 mM Tris HCl, pH 7.4, separated by SDS/PAGE, electrophoretically transferred to nitrocellulose, and probed for the presence of syntaxin, synaptobrevin, SNAP-25, or rab3A by Western blotting as indicated. Left lane, total eluate from rbSecl-GST beads; center lane, total eluate from GST beads; right lane, 0.4 mg of the Triton X-100 rat brain extract used as starting material.

reticulum to the Golgi complex and from the Golgi complex to the vacuole, respectively. Thus, Secl, Slyl, and Slpl represent a family of proteins that appear to play step-specific but homologous roles at different stations of the secretory pathway. Recently, proteins have been identified in D. melanogaster (Rop) and in C. elegans (UNC18) with sequence similarity to Secl that are expressed in the nervous systems of these organisms. The identification of these proteins has been the premise for our use of a PCR-based approach to identify a rat brain Secl homologue. Sequence comparisons suggest that rbSecl, as well as UNC18 and Rop, are slightly more related to Secl than to Slyl and Slpl. They are, therefore, likely to have a role in the late stages of the secretory pathway. This hypothesis is supported by additional experimental evidence in the case of Rop and UNC18. The Rop gene is expressed in Drosophila cells specialized for secretion (neurons, salivary glands) or exo/endocytotic recycling at the cell surface (garland cells) (25). In C. elegans, UNC18 is expressed at a very high concentration in neurons, and mutations in this gene cause phenotypes suggestive of an impairment of neurotransmitter secretion (27). It was reported that the secl-J yeast mutant strain is defective both in exocytosis and in endocytosis (45). The phenotypes of unc-18 C. elegans mutants, which lead to impaired neurotransmitter secretion, would be, in principle, consistent with a selective impairment in synaptic vesicle exocytosis, a selective impairment of endocytosis, or both. However, the accumulation of acetylcholine in the nervous system typical of these mutations suggests an accumulation of neurotransmitter-containing vesicles and, therefore, a block in exocytosis. A simple and unifying interpretation of the genetic data is that Secl and its homologues are selectively involved in exocytosis and reduce endocytosis by blocking recycling. The yeast syntaxin homologues Ssol and Sso2 were identified as the proteins encoded by high copy suppressors of a temperature-sensitive allele of SEC] (secl-J). Furthermore, it was shown that the SSO genes could not rescue yeast strains harboring a null mutant of SEC] (10), indicating that

Cell Biology: Garcia et A the Sso proteins do not replace the function of Secl but assist in the function of the temperature-sensitive mutant Secl protein. Our finding that rbSecl interacts with syntaxin strongly suggests that this rescue is mediated by the binding of Ssol and Sso2 to Secl and that, in general, the interaction of proteins of the Secl family with proteins of the Sso/ syntaxin family is required for exocytosis to occur. It can be speculated that similar interactions may take place at another step of the secretory pathway where not only Secl homologues (Slyl and Slpl in yeast) but also syntaxin homologues [Sed5 (46) and Pepl2 (GenBank accession no. M90395) in yeast and syntaxin 5 in rat (47)] have been implicated. A significant pool of brain syntaxin was found to form a complex with SNAP-25 and synaptobrevin in a Triton X-100solubilized brain extract (8). The recovery of syntaxin, but not of SNAP-25 and synaptobrevin, on immobilized rbSeclGST raises the possibility that only the pool of syntaxin not involved in a multimeric SNARE complex binds to Secl. This may explain the relatively low recovery of syntaxin on rbSecl-GST beads (unpublished observation). The stoichiometry of syntaxin binding to rbSecl remains to be investigated. It will be of interest to determine whether binding of Secl to syntaxin precedes formation ofthe SNARE complex. Genetic interactions have been reported between proteins of the Secl family and proteins of the Sec4/Yptl/rab family (24, 43). Although rab3a did not bind the rbSecl-GST protein, a role for Secl family proteins acting downstream to rab protein function in the sequence of events leading to membrane fusion is consistent with yeast data. What may be the significance of the nearly unique neuronspecific expression of rbSecl, UNC18, and Rop when yeast studies suggest that Secl plays an essential role in secretion? By Western blotting, the protein was undetectable in most nonneuronal tissues and was only barely detectable in some endocrine tissues (data not shown). Likewise, the rbSecl cDNA probe did not hybridize to other messages in nonneuronal tissues even when tested under intermediate stringency conditions. These findings may have two explanations. First, there is only one member of the Rop/UNC18/Secl family in the rat, and this gene is expressed at much higher concentrations in the nervous system than in other tissues because of high specialization of these cells for secretion. The other more likely possibility is that one or more related genes do exist in the rat but are sufficiently divergent so that under the conditions we tested the corresponding messages do not hybridize to clone 16 cDNA. It can be anticipated that further characterization of the Secl protein family will lead not only to identification of other mammalian Secl isoforms involved in exocytosis but also to identification of other proteins more closely related to Slyl and Slpl, which function in other steps of the secretory pathway. We thank Drs. S. Keranen, P. Novick, S. Ferro-Novick, P. McPherson, T. Galli, and M. Fortini for advice and discussion and L. Daniels and R. Dirkx for technical help. In addition, we thank Dr. C. Barnstable and R. Jahn for the generous gift of antibodies. This work was supported by grants from the National Institutes of Health (AI 30248, CA46128) and by a McKnight Research Project Award to P.D.C., by a fellowship from the National Institutes of Health (1 F32 NS09560-01) to E.P.G., and from the Associazione Italiana per la Ricerca sul Cancro to E.G. 1. Sudhof, T. C., De Camilli, P., Niemann, H. & Jahn, R. (1993) Cell 75, 1-4. 2. Bennett, M. K. & Scheller, R. H. (1993) Proc. Nat!. Acad. Sci. USA 90, 2559-2563. 3. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. & Rothman, J. E. (1993) Nature (London) 362, 318-324.

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