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 (//VAMP//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 (receivedfor review November 19, 1993)

ABSTRACT Secl is a hydrophilic protein that plays an factors required for fusion including N-ethylmaleimide- essential role in exocytosis from the yeast Saccharomyces sensitive fusion proteins, a-, /-, and y-SNAPs (3). Corre- cerevisiae. Two high copy suppressors of mutations in the Secl sponding yeast homologues, which play a general role in gene, SSOI and SS02, were recently identified that encode membrane fusion in the secretory pathway, have also been proteins of the syntaxin family. Syntain (a T-SNARE), to- identified (15-17). gether with SNAP-25 and synaptobrevin/VAMP (a T- and a It is quite clear, however, that additional components must V-SNARE, respectively), is thought to form the core of the be involved in the fusion machinery. For example, work in docking-fusion complex in synaptic vesicle exocytosis. Proteins yeast has defined several gene products that participate in that exhibit similarity to Secl were identified in the nervous exocytosis and for which there are no identified mammalian system of Drosophila melanogaster (Rop) and Caenorhabdiis homologues (18). One such protein is Secl (19, 20). Secl has ekgans (UNC18). Based on the amino acid sequence alignment sequence similarity to two other yeast proteins, Slpl (VPS33) ofSecl, Rop, and UNC18, we have used a PCR-based approach and Slyl, which are thought to operate in vesicular traffic to isolate a rat brain cDNA encoding a Secl homologue. The from the Golgi complex to the vacuole (21, 22) and from the cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern endoplasmic reticulum to the Golgi complex (23, 24), respec- blot analysis and encodes a protein of593 amino acids (rbSecl). tively. Two proteins that share sequence similarity with Secl Antibodies raised against a central portion of rbSecl recognize have been recently identified in Drosophila melanogaster a 67.5-kDa protein in total homogenates of rat brain but not of and in Caenorhabditis elegans. The Drosophila Secl homo- nonneuronal tissues. When incubated with a Triton X-100 logue, called Rop (Ras opposite), was identified as the protein brain extract, rbSecl-glutathione S-transferase (GST) fusion encoded by a gene that is localized next to the RAS2 gene and protein, but not GST protein alone, specificaly interacts with is transcribed in the opposite direction but under the control syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We of the same promoter. Rop is expressed in the nervous conclude that the function of proteins of the Secl family in system and in selected tissues with high secretory and membrane fusion involves an interaction with a T-SNARE. endocytic activity (25). The C. elegans Secl homologue, UNC18, is a protein whose mutations lead to a phenotype Elucidation of the molecular mechanisms by which synaptic consistent with an impairment of presynaptic function. vesicles dock and fuse with the plasmalemma has recently UNC18 was found to be expressed selectively in the nervous been the focus of intense investigation (1). Increasing evi- system (26-28). dence suggests that molecular mechanisms of synaptic ves- It appears, therefore, that Secl-related proteins may define icle exocytosis are fundamentally similar to mechanisms that a family of proteins that play an essential role in membrane operate in all types of exocytosis and, more generally, in all docking and/or fusion, including the exocytosis of synaptic membrane fusion events in the secretory and endocytic vesicles. Elucidation of the function of these proteins awaits pathway (2). the study of their biochemical interactions. The recent dem- A set of components that appear to form the core of the onstration that SS01 and SS02, two yeast genes encoding docking and fusion machinery for synaptic vesicles has been syntaxin homologues, are high copy suppressors of muta- identified. According to a recently proposed model, referred tions in the SEC] gene (10) suggests the attractive possibility to as the SNARE hypothesis (3), the synaptic vesicle protein that proteins of the syntaxin family interact directly with synaptobrevin/VAMP (referred to as a V-SNARE) (4, 5) proteins of the Secl family. The goal of this study was to interacts with syntaxin (6) and SNAP-25 (referred to as identify a rat brain homologue of Secl and to determine T-SNARES) (7), two proteins localized in the plasmalemma whether this with (3, 8). (VAMP is a vesicle-associated ; protein interacts syntaxin.t SNAP is a soluble N-ethylmaleimide-sensitive attachment protein fusion protein; and SNARE is a SNAP receptor.) MATERIALS AND METHODS Homologues of each of these proteins are present in yeast, and genetic analysis has demonstrated their participation in Cloning and Sequencing of rbSecl cDNA. Two degenerate exocytosis (refs. 9-11; V. Bankaitis, personal communica- oligonucleotide primers (AHVFFT and DAEGE, corre- tion). Furthermore, the crucial role of these proteins in sponding to amino acids 113-118 and 386-390 of Rop, re- neuronal exocytosis is emphasized by the demonstration that spectively) were synthesized based on the protein sequence they are targets for the proteolytic action of clostridial alignment of Rop, UNC18, and Secl (W. M. Keck Founda- neurotoxins, which are potent inhibitors of neurotransmitter tion Biotechnology Resource Laboratory, Yale University). release (12-14). Formation of the SNARE complex is then PCRs were carried out using 0.2 gg of a rat brain cDNA thought to be followed by the recruitment ofseveral cytosolic Abbreviations: rbSecl, rat brain Secl; GST, glutathione S-transfer- ase. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. GenBank data base (accession no. U06069). 2003 Downloaded by guest on September 25, 2021 2004 Cell Biology: Garcia et al. Proc. Nati. Acad. Sci. USA 91 (1994) library (29). A DNA product of the expected size was Eluates were separated from the beads by centrifligation at amplified (860 bp). This product was subcloned into pBlue- 13,000 x g and processed for SDS/PAGE and Western script II SK- (Stratagene), sequenced by standard methods blotting. (30, 31), and found to encode a sequence related to SEC]. The Nu_1enamProcedues. Tissues were homogenized and PCR fragment was radiolabeled with [yt32P]dCTP (Amer- processed for SDS/PAGE as described (35). SDS/PAGE and sham) by random priming (Boehringer Mannheim) and used Western blotting were performed essentially as described by to screen an oligo(dT)-primed A ZAP II rat brain cDNA Laeinmli (36) and Towbin et al. (37). Antibodies coupled to library (Stratagene, no. 936501). Twenty-five nitroceliulose alkaline phosphatase were used as secondary reagents for filters containing 20-30 x 103 phage plaques were screened Western blotting. (31). Fourteen putative positive clones were isolated and three plaques remained positive through sequential plaque purifications. Clones were subcloned into pBluescript II RESULTS SK- plasmid (Stratagene) and sequenced in both directions Degenerate PCR primers corresponding to amino acid se- by standard methods (30, 31). quences conserved among Secl, UNC18, and Rop were used Northern Blot Analyts. Total RNA was isolated from to amplify aportion ofarat brain cDNA (860 bp) that encoded tissues by the guanidine isothiocyanate procedure (32). a sequence related to Secl (see Materials andMethods). This Poly(A)+ RNA was isolated by oligo(dT)-cellulose and run on fragment was used to screen a A ZAP II rat brain cDNA formaldehyde agarose gels (32). The RNA was transferred to library (Stratagene). Three positive clones were isolated, Zeta-Probe nylon membrane (Bio-Rad) for 2 hr at 0.8 A in subcloned into pBluescript II SK-, and found to be 2.3 TAE (40 mM Tris acetate/2 mM EDTA, pH 8.0). The blot (clone 16), 1.8 (clone 24), and 3.0 (clone 22) kb long. Clones was hybridized for 16'hr at 3rC with [y-32P]dCTP random- 16 and 24 were sequenced in their entirety. The deduced primed clone 16 cDNA in 50% formamide/5x SSC (150 mM amino acid sequence ofclone 16 is shown in Fig. 1. It encodes NaCl/15 mM trisodium citrate, pH 7.0)/160 mM potassium a methionine at nucleotide position 18. The co ding phosphate, pH 7.4/1x Denhardt's reagent/50 pug of salmon codon is embedded in the sequence AACGCCATG that fits sperm DNA per ml. High stringency washes were performed the consensus defined by Kozak for an initiation codon (38). at 680C in 0.2x SSC/O.1% SDS. Eight of 10 nucleotides fit the consensus and. the two posi- Atibodies. A rabbit anti-rbSecl antiserum was obtained tions most critical for function (positions 4 and 10) are by injecting a recombinant protein composed of maltose present. This methionine is followed by a sgle 1781-lbp open binding protein (New England Biolabs) fused to amino acids reading frame that terminates at position 1797 encdig a 102-255 of rbSecl (see below). A rabbit anti-SNAP-25 anti- putative protein of 593 amino acids with a prdicted. Mr of serum was raised by using the protocol described by Qyler et 67,450, which we refer to as rbSecl. al. (7). The following anibdies were generous gifts: a mouse Clone 24 starts at position 23 of clone 16 and is identical to monoclonal antibody directed against syntaxin from C. Barn- it until position 1718-i.e., 81 bases upstream of its stop stable (Yale University) (33), a rabbit anti-rab3A antiserum codon. Beyond this position, clone 24 continues for an (34), and a monoclonal antibody directed against synapto- additional 45 bases, which have no homology to the corte- brevin (clone 10.3) (4) from R. Jahn (Yale University). sponding region of clone 16. Given the evidence for a single Produton of rbSecl Fuion Proteins. Clone 16 was sub- mRNA species (see below), this divergent region ofdione 24 cloned into the bacterial expression vector pGEX-2T (Phar- may represent a cloning artifact although the possiiity that macia) obtaining an-in-frame recombinant protein composed it may represent an alternatively spliced form cannot be ofglutathione S-transferase (GST) fused to the N terminus of excluded at this time. Restriction analysis and partial se- rbSecl. rbSecl-GST fusion protein and GST were produced quence of clone 22, which start at position 330 of clone 16, in Escherichia coli, and soluble bacterial extracts containing confirhed the sequence of clone 16. rbSecl-GST fusion protein or GST protein were then affin- The amino acid sequence ofthe rbSecl exhibits a substan- ity-purified on glutathione-Sepharose 4B beads (Pharmacia) tial similarity along its entire length to other proteins of the (32). Another fusion protein composed ofthe maltose bindi Seci family (Fig. 1). As shown in Table 1, rbSecl is most protein fused to amino acids 102255 ofrbSecl was produced similar to Rop (25) (65%) and increasingly less similar to in the pMAL-p expression vector (New England Biolabs). UNC18 (27) (59o), Secl (39) (27%), Slyl (22) (22%), and SIp1 The cDNA encoding the rbSecl portion of the fusion protein (21) (20%). This hierarchy of similarity agrees with evolu- was obtained by PCR using synthetic oligonucleotide primers tionary considerations and is consistent with a role ofrbSecl flanked by BamHI and Xba I cleavage sites. RbSecl protein in-exocytosis. The overall size ofall the proteins ofthe family present in soluble bacterial extracts was affinity-purified on is very similar and only Secl has an additional C-terminal amylose resin (New England Biolabs) and used to immunize extension. The N terminus of rbSecl aligns well with the N rabbits. terminus ofRop UNC18 and Secl. Thisfinding, together with Binding of rb&ecl-GST to Triton X-1O-WSolubillzed Rat the presence of a strong Kozak consensus (see above), Brain Exrac. Frozen rat brains (Pel-Freez Biologicals) supports the correct assignment of the start methionine in were homogenized (1:20, wt/vol) in 150 mM NaCl/10 mM clone 16, the clone with the longest 5' extension, in spite of Hepes-KOH, pH 7.4, containing pepstatin, aprotinin, leu- the lack of any stop codon in the short nucleotide sequence peptin, antipain (each at 4 pg/ml), 0.4 ;M phenylmethylsul- preceding this methionine. fonyl fluoride, and 10 mM benzamidene using an SDT Salzberg et al. (25) identified a a-Cop motif in a 27-amino Tissumjzer (Tekmar, Cincinnati). Triton X-100 (Sigma) was acid stretch of Rop, Secl, Slyl, and Slpl. This motif is also then added, to a final concentration of 0.5% (vol/vol); the present in rbSecl and in UNC18. Like other members of the mixture was incubated at 40C for 1 hr with constant agitation family, rbSecl is predicted to be a hydrophilic protein (pI and subsequently clarffied by centrifugation at 200,000 x g 7.01), with a high content (311%) of charged amino acids and for'2 hrat 40C. Glutathione-Sepharose 4B beads preincubated no putative transmembrane regions. The Swiss-Prot and the with similar amounts of RbSecl-GST or GST were added to GenBank/EMBL data bases were searched for additional 1 ml of the supernatant and incubated with gentle mixing for related sequences at the protein and nucleotide levels, re- 2 hr at 40C. Beads were then washed three times in ice-cold spectively. The only substantial homology was found be- 150mM NaCl/10 mM sodium phosphate, pH 7.4, with gentle tween the 3' end of clone 16 and the nucleotide sequence of mixing for 10 min and eluted with 10mM Tris HCl, pH 7.4/5 a human' brain cPNA (GenBank. accession no. M79125) (40). mM glutathione (Sigma) for 30 min with gentle mixing. The sequence of this partial cDNA contains at its 5' end an Downloaded by guest on September 25, 2021 Cell Biology: Garcia et al. Proc. Natl. Acad. Sci. USA 91 (1994) 2005 1 _ _ 85 rbSecl Rop N. YKPPPP KKQTSAAGAGG LVV RKL H EGITMVEDIN REPLPT Unc18 M..D.ILKL RN SGcR.K IS Secl NKTVETI PQELLN NUS D LIJDSPT 86 170 rbSecl LE YLITPSEPSE HSIS VKFPSKY VjIKTLTEINIAFLPYESQY[ .LD S Rop IYLITPSD S G L P HVFF E LF .KSAAGIKTLKEINIAFLPYEjQ IF.ILDSIMDLP

Unc18 LE IY P A 1 KLI Y. ..CARN HVFF A LT. R IKTLKEINIA S F N .L Secl E IJILEITKYNINCIDA MVI43. KR LPGJlNPIEQFFQS NLRYEISQNLESESEFFE IIQMEIL 171 255

rbSecl KS MKNPI LAEQIATLCAT RYEYK AL...... I KLDAYKADDPT Rop MC1|Y SIRSKH IAEQIATLCAT RY SDWD ID ...... L SA KLDAYKAD Unc18 FLY GGLTS EIAEQI CAT RYDFE VE...... LDAYKADDPtSA Secl QV1FNNNCKALIPTNVRKIVGSLV SVSNPVEEEDARNGNAVVNANSLTRSI NA fIAI DFPP 251 340 rbSecl EGP K RSQLL ILDRGFDP|SpLHEL QA IENDVYKYESIRE KE. LLD LRHKHI Rop EGP uK RSQLLILDRGFD CVSP LHEL LQA YDLL IMNDVY YG. .PNQP KEV.. .LLD LRHI Uncl8 S KARSO Q~IIDRD AI MP LHEL QA YDL ... IENDVYKY SDNL KEV. LLD EREHKHIhJMV Secl QNT II lp I ~ Y QAIHSNVD AENEAGEQE XS DAN

3 4 1 _4 2 5 rbSec1 EVT SSK RDLQMLKKMPQYQKELSKYSTHLHLAEDCIM. GDKLCRVEQDL GTDAEG Rop T QL DS..GSAD S KKMPQYQKELSKYSTHLHLAED VDKLCRVEQDL AEGE Unc18 ESKGN GE3MDSKS DLD PH E ST I LA C . DKLQ EQD S. IGIIDAEGE Secl EYIQGRIL LIAKNPLLVDRSNVKNT KSVVAHLKDFDERRRLK ENAERKLADISAI S G 426 -_510 rbSecl |KIK P RIILLYLFLKNGITEENLNKI VP LRR Rop JKIK IVPILLDANV RIIAL MIIKNGIfEENflK-LF HA L QD ...... My Unc18 RV AKL PL ILLYILS KNGI MENLNK4A.LGLLEITGRKKTW Seci1 |K I K|H I I DDL tIJAMKEP T I YII LFRqIfLDFIUKLNFIGVTHHENFQQYLIF~f YDLIDFKLI I4KPKDK.. 571 595 rbSecl KDfRK ERT...... W MEIlED.IK.| LDTKH Y ISTfSSASFSTTAVAG.H Rop VPI.KEFTTE...... SRWTPVIKDIME lEDE ..LDH|FAP|FLERAQNTNYHAPT |RYG.L . ... WHID Unc18 PT KqKE8PIEH...... SYM|SII ..LDTKH F LA QVNQGY RAP ARQ ...... HXE Secl1 .FKWFHDTLVNDPNI HISF VGLSK INPLLJSEQYF1LKDKPIELLNEEEFQGLANTSANSSSSLRNP

596 680 rbSec1 lqA. PGEYR EMRCAYEV EVLIG HTS EISS*End Rop .4.LG .QAQVKNV ... PRLIfFITGGV SEMRCAYEV R EV LG HIIf EI I SLSjG E4L4End ...... Uncl8 ..RGQQSNffjK..... RLII IIGG TFS IGSVDKIPEDfFK LR E.nPRD Sec1 AWTTKSSNIKKNIPRFFYYVI IIP QN .LKNRDIFIGSD IL P TF E E PFFKFKEDQRQQVNP

681 765 rbSecl ......

Rop ...... Uncl18 ...... Secl PDFLLREMKPVAQPVSHVHLKSQDNSPKSGTSSPKAAGSLKSEPPEKEKKRSKFSRFLKRKSHHDK*End

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 ofRop (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 iden- were observed in other tissues (data not shown). An antise- tical to the last 24 amino acids of rbSecl. These 24 amino rum raised against rbSecl (but not the preimmune serum) acids are followed by a termination codon in both sequences. recognized a protein band with an apparent molecular mass This observation strengthens the conclusion that the C ter- minus assigned to rbSecl by the sequence of clone 16 is correct. < a m QI -dI m Northern blots of rat poly(A)+ mRNA from brain, heart, z > c -0 kidney, liver, and lung were probed with [y-32P]dCTP-labeled I I I I I full-length clone 16 to determine the size and tissue distri- -9.49 kb bution of the rbSecl mRNA. Under high stringency, we -7.46 kb detected a single message of 3.5 kb selectively expressed in brain tissue (Fig. 2), indicative of a single rbSecl-related -4.40 kb mRNA. Only after prolonged exposure ofthe autoradiogram, I- extremely faint bands with the same electrophoretic mobility -2.37 kb Table 1. Sequence similarities among Secl-related proteins rbSecl Rop Uncl8 Secl Slyl Sipi -1.35 Kb rbSecl - 0.65 0.59 0.27 0.22 0.20 Rop 0.65 0.59 0.27 0.22 0.20 Unc18 0.59 0.59 0.26 0.23 0.21 FIG. 2. Northern blot analysis of rat tissues. Three micrograms Secl 0.27 0.27 0.26 0.24 0.20 ofpoly(A)+ mRNA from the indicated tissues or rRNA (isolated from Slyl 0.22 0.22 0.23 0.24 0.19 lung) was applied to each lane. The blot was probed with random- SIpi 0.20 0.20 0.21 0.20 0.19 primed [-y-32P]dCTP-labeled clone 16 cDNA and washed under high stringency conditions as described. Downloaded by guest on September 25, 2021 2006 Cell Biology: Garcia et al. Proc. Natl. Acad. Sci. USA 91 (1994)

of 67 kDa in a brain extract probed by Western blotting (Fig. +-d 3A). This size is in very good agreement with the predicted 4J-- x molecular mass ofthe protein (see above). This band was not LU detected in extracts ofpituitary, pancreas, testis, heart, lung, O kidney, liver, PC12 cells (41), and rat insulinoma cells CD (RINm5) (42) (Fig. 3B). H- We next investigated whether recombinant rbSecl could C- a) .-t interact with syntaxin. A GST fusion protein that contains the 0I- I- entire open reading frame of rbSecl was constructed in I-r pGEX2T vector. The apparent molecular mass ofthe protein in SDS/PAGE was 95 kDa as predicted by the coding "new -Syntaxin sequence. Furthermore, rbSecl-GST protein was highly immunoreactive with anti-rbSecl antibody (data not shown). rbSecl-GST and GST immobilized on glutathione-Sepharose -F-Synaptobrevin / VAMP 4B beads were incubated with Triton X-100-solubilized brain extract. Proteins specifically bound to the beads were then _,- SNAP-25 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 - rab3A shown by Fig. 4, syntaxin, but not synaptobrevin/VAMP or SNAP-25, was specifically recovered on the rbSecl-GST FIG. 4. Biochemical interaction of rbSecl with syntaxin. Equal beads and not by the GST beads. Genetic analysis of sec amounts of rbSecl-GST or GST coupled to glutathione-Sepharose mutant yeast strains suggests that the SEC] and SLY] genes 4B beads were incubated with 10 mg ofa Triton X-100-solubilized rat may interact with the SEC4 and YPTI genes, respectively brain extract. Specifically bound proteins were eluted in 5 mM (24, 43). Because YPTI and SEC4 are members of the glutathione/10 mM Tris HCl, pH 7.4, separated by SDS/PAGE, electrophoretically transferred to nitrocellulose, and probed for the family (44), we probed the material eluted from rbSecl-GST presence of syntaxin, synaptobrevin, SNAP-25, or rab3A by West- beads with antibodies directed against rab3A, a rab protein ern blotting as indicated. Left lane, total eluate from rbSecl-GST concentrated on synaptic vesicles (34). No rab3A was de- beads; center lane, total eluate from GST beads; right lane, 0.4 mg tected. of the Triton X-100 rat brain extract used as starting material. reticulum to the Golgi complex and from the Golgi complex DISCUSSION to the vacuole, respectively. Thus, Secl, Slyl, and Slpl Here we describe the identification of a rat brain protein, represent a family ofproteins that appear to play step-specific rbSecl, similar to the yeast Secl protein and demonstrate but homologous roles at different stations of the secretory that rbSecl interacts selectively with syntaxin, one of the pathway. Recently, proteins have been identified in D. mel- SNAREs involved in neurosecretion. This finding provides anogaster (Rop) and in C. elegans (UNC18) with sequence additional information regarding the molecular mechanisms similarity to Secl that are expressed in the nervous systems involved in synaptic vesicle exocytosis. In addition, this of these organisms. The identification of these proteins has result is likely to have implications for the elucidation of been the premise for our use of a PCR-based approach to mechanisms of vesicle docking and fusion in other types of identify a rat brain Secl homologue. Sequence comparisons exocytosis and in vesicular traffic at other stations of the suggest that rbSecl, as well as UNC18 and Rop, are slightly secretory pathway. more related to Secl than to Slyl and Slpl. They are, Seci is a protein that participates selectively in exocytosis therefore, likely to have a role in the late stages of the from yeast (19), while two related yeast proteins, Slyl (22) secretory pathway. This hypothesis is supported by addi- and Slpl (21), are involved in transport from the endoplasmic tional experimental evidence in the case of Rop and UNC18.

C . The Rop gene is expressed in Drosophila cells specialized for A secretion (neurons, salivary glands) or exo/endocytotic re- C-E cycling at the cell surface (garland cells) (25). In C. elegans, UNC18 is expressed at a very high concentration in neurons, E and mutations in this gene cause phenotypes suggestive ofan impairment of neurotransmitter secretion (27). It was reported that the secl-J yeast mutant strain is rbSec 1- defective both in exocytosis and in endocytosis (45). The phenotypes of unc-18 C. elegans mutants, which lead to U) impaired neurotransmitter secretion, would be, in principle, B MI m U) consistent with a selective impairment in synaptic vesicle N 4 _J- >, C t exocytosis, a selective impairment of endocytosis, or both. LULU . U aa n CL CL I However, the accumulation of acetylcholine in the nervous c I l system typical of these mutations suggests an accumulation of neurotransmitter-containing vesicles and, therefore, a block in exocytosis. A simple and unifying interpretation of rbSec 1-- the genetic data is that Secl and its homologues are selec- tively involved in exocytosis and reduce endocytosis by blocking recycling. FIG. 3. Detection and tissue distribution of rbSecl. (A) Western The yeast syntaxin homologues Ssol and Sso2 were iden- blot of a brain extract (100 tg of total protein per lane) was probed with anti-rbSecl antiserum or preimmune serum, both diluted to tified as the proteins encoded by high copy suppressors of a 1:100. (B) Two hundred micrograms ofeach tissue tested was loaded temperature-sensitive allele of SEC] (secl-J). Furthermore, per lane and Western blotted (as indicated) with anti-rbSecl anti- it was shown that the SSO genes could not rescue yeast serum. strains harboring a null mutant of SEC] (10), indicating that Downloaded by guest on September 25, 2021 Cell Biology: Garcia et A Proc. Natl. Acad. Sci. USA 91 (1994) 2007 the Sso proteins do not replace the function ofSecl but assist 4. Baumert, M., Maycox, P. R., Navone, F., De Camilli, P. & Jahn, R. in the function of (1989) EMBO J. 8, 379-384. the temperature-sensitive mutant Secl 5. Trimble, W. S., Cowan, D. M. & Scheller, R. H. (1988) Proc. Natl. protein. Our finding that rbSecl interacts with syntaxin Acad. Sci. USA 85, 4538-4542. strongly suggests that this rescue is mediated by the binding 6. Bennett, M. K., Calakos, N. & Scheller, R. H. (1992) Science 257, of Ssol and Sso2 to Secl and that, in general, the interaction 255-259. 7. Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, of proteins of the Secl family with proteins of the Sso/ M., Bloom, F. E. & Wilson, M. C. (1989) J. Cell Biol. 109, 3039-3052. syntaxin family is required for exocytosis to occur. It can be 8. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. & speculated that similar interactions may take place at another Rothman, J. E. (1993) Cell 75, 409-418. 9. DeCamilli, P. (1993) Nature (London) 364, 387-388. step of the secretory pathway where not only Secl homo- 10. Aalto, M. K., Ronne, H. & Keranen, S. (1993) EMBO J. 12, 4095-4104. logues (Slyl and Slpl in yeast) but also syntaxin homologues 11. Protopopov, V., Govindan, B., Novick, P. & Gerst, J. E. (1993) Cell 74, [Sed5 (46) and Pepl2 (GenBank accession no. M90395) in 855-861. yeast and syntaxin 5 in rat (47)] have been implicated. 12. Schiavo, G., Rossetto, O., Santucci, A., DasGupta, B. R. & Monte- cucco, C. (1992) J. Biol. Chem. 267, 23479-23483. A significant pool of brain syntaxin was found to form a 13. Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, complex with SNAP-25 and synaptobrevin in a Triton X-100- P., Sudhof, T. C., Niemann, H. & Jahn, R. (1993) Nature (London) 365, solubilized brain extract (8). The recovery of syntaxin, but 160-163. 14. Blasi, J., Chapman, E. R., Yamasaki, S., Binz, T., Niemann, H. & Jahn, not of SNAP-25 and synaptobrevin, on immobilized rbSecl- R. (1993) EMBO J. 12, 4821-4828. GST raises the possibility that only the pool of syntaxin not 15. Whiteheart, S. W., Griff, I. C., Brunner, M., Clary, D. O., Mayer, T., involved in a multimeric SNARE complex binds to Secl. Buhrow, S. A. & Rothman, J. E. (1993) Nature (London) 362, 353-355. This may explain the relatively low recovery of syntaxin on 16. Wilson, D. W., Whiteheart, S. W., Wiedmann, M., Brunner, M. & Rothman, J. E. (1992) J. Cell Biol. 117, 531-538. rbSecl-GST beads (unpublished observation). The stoichi- 17. Kaiser, C. A. & Schekman, R. (1990) Cell 61, 723-733. ometry of syntaxin binding to rbSecl remains to be investi- 18. Pryer, N. K., Wuestehube, L. J. & Schekman, R. (1992) Annu. Rev. gated. It will be of interest to determine whether binding of Biochem. 61, 471-516. Secl to syntaxin precedes formation ofthe SNARE complex. 19. Novick, P., Field, C. & Schekman, R. (1980) Cell 21, 205-215. 20. Aalto, M. K., Ruohonen, L., Hosono, K. & Keranen, S. (1991) Yeast 7, Genetic interactions have been reported between proteins of 643-650. the Secl family and proteins ofthe Sec4/Yptl/rab family (24, 21. Wada, Y., Kitamoto, K., Kanbe, T., Tanaka, K. & Anraku, Y. (1990) 43). Although did not bind the rbSecl-GST protein, a Mol. Cell. Biol. 10, 2214-2223. role for Secl family proteins acting downstream to rab 22. Robinson, J. S., Klionsky, D. J., Banta, L. M. & Emr, S. D. (1988) Mol. Cell. Biol. 8, 4936-4948. protein function in the sequence of events leading to mem- 23. Ossig, R., Dascher, C., Trepte, H. H., Schmitt, H. D. & Gallwitz, D. brane fusion is consistent with yeast data. (1991) Mol. Cell. Biol. 11, 2980-2993. What may be the significance of the nearly unique neuron- 24. Dascher, C., Ossig, R., Gallwitz, D. & Schmitt, H. D. (1991) Mol. Cell. specific expression of rbSecl, UNC18, and Rop when yeast Biol. 11, 872-885. 25. Salzberg, A., Cohen, N., Halachmi, N., Kimchie, Z. & Lev, Z. (1993) studies suggest that Secl plays an essential role in secretion? Development 117, 1309-1319. By Western blotting, the protein was undetectable in most 26. Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishi- nonneuronal tissues and was only barely detectable in some waki, K., Miwa, J., Taketo, A. & Kodaira, K. I. (1992) J. Neurochem. endocrine tissues (data not shown). Likewise, the rbSecl 58, 1517-1525. 27. Gengyo-ando, K., Kamiya, Y., Yamakawa, K., Kodaira, K., Nishiwaki, cDNA probe did not hybridize to other messages in nonneu- K., Miwa, J., Hori, I. & Hosono, R. (1993) Neuron 11, 703-711. ronal tissues even when tested under intermediate stringency 28. Pelham, H. R. B. (1993) Nature (London) 364, 582. conditions. These findings may have two explanations. First, 29. Burton, J., Roberts, D., Montaldi, M., Novick, P. & De Camilli, P. (1993) there is only one member of the Rop/UNC18/Secl family in Nature (London) 361, 464-467. 30. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., the rat, and this gene is expressed at much higher concen- Fiddes, C. A., Hutchison, C. A., Slocombe, P. M. & Smith, M. (1977) trations in the nervous system than in other tissues because Nature (London) 265, 687-695. of high specialization of these cells for secretion. The other 31. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A more likely possibility is that one or more related genes do Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY). 32. Ausubel, F. M., Brent, R., Kingston, R. I., Moore, D. D., Seidman, exist in the rat but are sufficiently divergent so that under the J. G., Smith, J. A. & Struhl, K. (1990) Current Protocols in Molecular conditions we tested the corresponding messages do not Biology (Wiley, New York). hybridize to clone 16 cDNA. It can be anticipated that further 33. Barnstable, C. J., Hofstein, R. & Akagawa, K. (1985) Brain Res. 352, characterization of the Secl protein family will lead not only 286-290. 34. Matteoli, M., Takei, K., Cameron, R., Hurlbut, P., Johnston, P. A., to identification of other mammalian Secl isoforms involved Sudhof, T. C., Jahn, R. & De Camilli, P. (1991)J. CellBiol. 115, 625-633. in exocytosis but also to identification of other proteins more 35. Cameron, P. L., Sudhof, T. C., Jahn, R. & De Camilli, P. (1991) J. Cell closely related to Slyl and Slpl, which function in other steps Biol. 115, 151-164. of the secretory pathway. 36. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 37. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nat!. Acad. Sci. USA 76, 4350-4354. We thank Drs. S. Keranen, P. Novick, S. Ferro-Novick, P. 38. Kozak, M. (1991) J. Cell Biol. 115, 887-903. McPherson, T. Galli, and M. Fortini for advice and discussion and 39. Aalto, M. K., Ruohonen, L., Hosono, K. & Keranen, S. (1992) Yeast 8, L. Daniels and R. Dirkx for technical help. In addition, we thank Dr. 587-588. C. Barnstable and R. Jahn for the generous gift of antibodies. This 40. Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, work was supported by grants from the National Institutes of Health J. M., Utterback, T. R., Nagle, J. W., Fields, C. & Venter, J. C. (1992) (AI 30248, CA46128) and by a McKnight Research Project Award to Nature (London) 355, 632-634. 41. Greene, L. A. & Tischer, A. S. (1976) Proc. Nat!. Acad. Sci. USA 73, P.D.C., by a fellowship from the National Institutes of Health (1 F32 2424-2428. NS09560-01) to E.P.G., and from the Associazione Italiana per la 42. Bhathena, S. J., Oie, H. K., Gazdar, A. F., Voyles, N. R., Wilkins, Ricerca sul Cancro to E.G. S. D. & Recant, L. (1982) Diabetes 31, 521-531. 43. Salminen, A. & Novick, P. (1987) Cell 49, 527-538. 1. Sudhof, T. C., De Camilli, P., Niemann, H. & Jahn, R. (1993) Cell 75, 44. Valencia, A., Chardin, P., Wittinghofer, A. & Sander, C. (1991) Bio- 1-4. chemistry 30, 4637-4648. 2. Bennett, M. K. & Scheller, R. H. (1993) Proc. Nat!. Acad. Sci. USA 90, 45. Riezman, H. (1985) Cell 40, 1001-1009. 2559-2563. 46. Hardwick, K. J. & Pelham, H. R. B. (1992) J. Cell Biol. 119, 513-521. 3. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., 47. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Geromanos, S., Tempst, P. & Rothman, J. E. (1993) Nature (London) Fleming, A. M., Hazuka, C. D. & Scheller, R. H. (1993) Cell 74, 362, 318-324. 863-873. Downloaded by guest on September 25, 2021