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Home , SNARE (protein), Synaptobrevin, Syntaxin

Journal of Cell Science 113, 4055-4064 (2000) 4055 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1894

SNAP-24, a SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands

Barbara A. Niemeyer*,‡ and Thomas L. Schwarz§ Department of Molecular and Cellular Physiology, Stanford Medical School, Stanford, CA 94305, USA *Present address: Department of Pharmacology and Toxicology, School of Medicine, University of Saarland, D-66421 Homburg, Germany ‡Author for correspondence (e-mail: [email protected]) §Present address: Harvard Medical School, Division of Neuroscience, The Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA

Accepted 16 September; published on WWW 31 October 2000

SUMMARY

Fusion of vesicles with target membranes is dependent is not concentrated in synaptic regions. In vitro studies, on the interaction of target (t) and vesicle (v) SNARE however, show that SNAP-24 can form core complexes with (soluble NSF (N-ethylmaleimide-sensitive fusion ) and both synaptic and non-synaptic v-SNAREs. attachment protein receptor) located on opposing High levels of SNAP-24 are found in larval salivary glands, membranes. For fusion at the plasma membrane, the t- where SNAP-24 localizes mainly to granule membranes SNARE SNAP-25 is essential. In Drosophila, the only rather than the plasma membrane. During glue secretion, known SNAP-25 isoform is specific to neuronal axons and the massive exocytotic event of these glands, SNAP-24 and additional t-SNAREs must exist that mediate containing granules fuse with one another and the apical both non-synaptic fusion in and constitutive and membrane, suggesting that glue secretion utilizes regulated fusion in other cells. Here we report the compound and that SNAP-24 mediates identification and characterization of SNAP-24, a closely secretion. related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP- Key words: SNARE, Vesicle, Specificity, Compound fusion, 24 in the nervous system is punctate and, unlike SNAP-25, Exocytosis, Drosophila

INTRODUCTION (Fasshauer et al., 1999; Yang et al., 1999). That the promiscuity of binding of different SNARE partners in vitro may not Vesicular transport is mediated by proteins present on the accurately reflect the specificity within a cellular system has vesicle membrane (such as /VAMP) and recently been demonstrated in a reconstituted PC12 secretion corresponding partners such as and SNAP-25 assay (Scales et al., 2000). (synaptosomal-associated protein of 25 KD) on the target Because the components of vesicular transport and synaptic membrane. The binding of synaptobrevin to plasma membrane transmission are well conserved across species, Drosophila syntaxin and SNAP-25 constitutes the core complex that is provides an excellent model system in which to study secretion essential for the fusion of vesicles. The importance of SNAP- and synaptic transmission by genetic, biochemical and 25 in membrane fusion has been demonstrated most directly physiological methods. Before the release of the Drosophila by the use of the clostridial A and genome sequence (Adams et al., 2000), the following E (BoNT/A or BoNT/E), which selectively cleave SNAP-25 homologues of the SNARE proteins necessary for vesicle and thereby block (Banerjee et al., 1996; trafficking to the plasma membrane had been identified: Lawrence et al., 1996). Other proteins may act to regulate or syntaxin 1 (syx) (Burgess et al., 1997; Parfitt et al., 1995; catalyze fusion by interacting with this core complex. These Schulze et al., 1995), SNAP-25 (Risinger et al., 1993; Risinger additional proteins include the N-ethylmaleimide sensitive et al., 1997) and synaptobrevin (syb and n-syb) (Chin et al., factor (NSF), an ATPase required for in vitro membrane fusion 1993; Deitcher et al., 1998; DiAntonio et al., 1993; Sudhof et during vesicular transport, and the soluble NSF-attachment al., 1989). The presence of two synaptobrevin homologues in proteins (α-, β-, γ-SNAPs) (Rothman and Wieland, 1996; Drosophila suggests that they might act as target-specific Sollner et al., 1993). The binding of a particular v-SNARE to determinants. One is a neuronal isoform (n-syb) and the other an appropriate t-SNARE partner has also been hypothesized to is generally expressed (syb). Expression of n-syb cleaving contribute to the specificity that is necessary to target vesicles toxin or disruption of the n-syb gene prevents action- to an appropriate membrane (Rothman and Wieland, 1996; potential-evoked release at the , though Sollner et al., 1993), but this specifying role is questionable, at spontaneous vesicle fusions persist (Deitcher et al., 1998; least when considered in biochemical experiments in vitro Sweeney et al., 1995; Yoshihara et al., 1999). In contrast, 4056 B. A. Niemeyer and T. L. Schwarz mutations in syb lead to cell death before the nervous system chromosomal location was determined by in situ hybridization of develops (S. Battacharya and T. L. Schwarz, unpublished biotinylated probes containing either full-length cDNA or P1 DNA to observations). If these v-SNAREs are specifying labels to polytene salivary gland chromosomes. target distinct classes of vesicle to separate portions of the cell, Sequence and mapping was recently confirmed by the Drosophila then t-SNAREs that distinguish among them should be present genome project. on the target membranes. However, Drosophila syntaxin 1A is Northern blot analyses ubiquitously expressed and is involved in both regulated Total RNA of Canton-S flies was prepared by homogenizing approx. vesicular fusion (e.g. synaptic transmission) and in constitutive 100 mg of tissue per ml Trizol reagent using a Dounce homogenizer. fusion (e.g. cellularization and tissue development) in most RNA was isolated following the manufacturer’s instructions (Gibco- (and perhaps all) cells (Burgess et al., 1997; Schulze et al., BRL). 20 µg of RNA were fractionated by electrophoresis on an 0.8% 1995). The only SNAP-25 gene known in Drosophila has a agarose-formaldehyde gel and transferred to a nitrocellulose strictly neuronal expression pattern (Risinger et al., 1993; membrane in 10× SSC. A [α-32P]GTP-labeled probe was prepared by Risinger et al., 1997). This observation suggests that more random priming of either the 1.6 kb cDNA insert or a 250 bp PCR ubiquitously expressed homologues exist that mediate fragment with low to SNAP-25. transport vesicle to plasma membrane fusion outside the Antibodies , and raises the possibility that those isoforms could To generate antibodies specific to SNAP-24, we synthesized peptides distinguish synaptic and non-synaptic vesicles within a given corresponding to regions of the protein that diverge from the SNAP- . 25 sequence: residues 126-142 (ERERGGMGAPPQSGYVA) called To address these questions of t-SNARE function outside the M24 and residues 193-208 (DANNIRMDGVNKRANN) called C24. synapse and the specifying potential of t-SNAREs for vesicle One additional was added at the N terminus to facilitate targeting, we have identified two novel members of the SNAP- crosslinking. C24 peptide was conjugated either to maleimide- 25 family in Drosophila. We tested the hypothesis that one activated keyhole limpet hemocyanin (KLH), following the suppliers new homologue, SNAP-24, and the known SNAP-25, may protocol (Pierce), or to glutaraldehyde-activated KLH. M24 peptide selectively form core complexes with syb and n-syb. We further was conjugated to glutaraldehyde-activated KLH (Mi et al., 1995), characterized SNAP-24 localization and the differential injected into rabbits and rats, and antiserum was affinity purified as distribution of SNAP-24 and SNAP-25. We found the salivary previously described (Mi et al., 1995). For immunoblot analysis, SNAP-24 antibodies were used at a 1:100 dilution in PBS, 5% dry glands to be a particularly rich source of SNAP-24 milk. When tested against fusion protein (see Results), two rabbit immunoreactivity and characterized its subcellular localization antibodies proved to be specific and are referred to as M24 and C24 and translocation during salivary gland secretion. (from maleimide-activated, KLH-injected rabbit). These antibodies were used for all further analyses. MATERIALS AND METHODS Immunohistochemistry Dissected whole salivary glands or brains from third instar larvae were cDNA cloning fixed in 3.7% formaldehyde in either PBS or a buffer containing 100 Two synthetic degenerate oligonucleotides with incorporated mM Pipes, pH 7.0, 2 mM EGTA, 1 mM MgSO4, for 20 minutes at restriction recognition sequences were used in a combined room temperature. Tissues were then washed at least three times for RT-PCR/PCR reaction (GeneAmp, Perkin Elmer): GGAAT- 30 minutes with phosphate-buffered saline (PBT: 1× PBS, 0.1% TCTTGATGAICA(A/G)GGIGA(I/A)CA(G/A)(C/T)T and CCAA- Triton X-100, 0.1% BSA) and blocked with PBT + 5% normal goat GCTTTTA(G/A)TT(T/C)TCITCCAT(C/T)TC(G/A)T(T/C)(C/T)TC, serum (PBT-NGS). SNAP-24 antibodies were diluted 1:100 or 1:50, corresponding to conserved amino acid (aa) sequences D(D/E)Q- anti-syntaxin antibody (8C3) 1:50, and anti-SNAP-25 antibody 1:100, (G/K)EQL and E(D/N)EM(E/D)EN, respectively, of SNAP-25 family in PBT-NGS solution, and samples were incubated with rotation members. The template cDNA was generated from poly(A)+ selected overnight at 4°C. In peptide-blocked control experiments, approx. 1 RNA isolated from 0-8 hour embryos using random hexamer primers mg/ml C24 antibody was preincubated overnight with 82 mg/ml in the RT-PCR reaction. peptide in PBS or approx. 0.5 mg/ml M24 antibody with 65 mg/ml A 200 bp PCR product was subcloned, sequenced and subsequently peptide. After incubation with secondary fluorescently labeled [α-32P]GTP labeled (High Prime, Boehringer Mannheim), then used antibodies for 1 hour at room temperature, tissues were mounted in as a probe to screen 800,000 plaques of the LD cDNA library (mRNA Vectashield (Vector Laboratories). The crossreactivity of 8C3 seen on source 0-22 hour embryos; Berkeley genome project). Hybridization, immunoblots of recombinant protein (see Fig. 4A) may be sufficiently rescreening and cDNA isolation was performed following standard weak not to have caused artifacts to date, as immunoblots of fly Stratagene cloning instructions. Automated cycle sequencing (ABI) extracts and immunohistochemistry of tissues show no crossreactivity was performed using both vector and internal primers and a single 1.6 with SNAP-24 and SNAP-25. Images were acquired and digitized kb cDNA was isolated that contained the 200 bp PCR fragment. using a confocal microscope (Molecular Probes) with pinhole Sequence data was analyzed using Omiga 2.0 software. Multiple openings of 50 or 100 µm. Laser intensity and photomultiplier (PMT) alignment was performed using the ClustalW program with the sensitivity were kept identical in experiments using the peptide- following parameters: open gap penalty=4, extend gap penalty=0.2, blocked primary antibody. Image analysis was performed using NIH delay divergent=60%, and some alignment was also done by eye. The Image and Adobe Photoshop software. Larval tissues were obtained putative SNAP-29 gene was found by searching the genome database from Oregon-R flies. for homologues of rat SNAP-29 and Drosophila SNAP-24. DNA constructs and production of recombinant proteins Chromosomal mapping GST-tagged Drosophila SNAP-25, SNAP-24, syntaxin 1A (aa 4-269), Full-length labeled cDNA was used to screen Drosophila high density n-syb (aa 1-104) and syb (aa 1-110) were constructed with PCR P1 filters (Genome Systems, Inc.). Four P1 clones were identified and primer sites for subcloning into pGEX-KG. Constructs were validated rescreened; DS04886 contained the SNAP-24 sequence, as confirmed by DNA sequencing. GST fusion proteins were expressed either in by sequencing. Because this P1 has not been mapped, the the AB1899 or the BL21 strain of Escherichia coli. Recombinant SNAP-24, a novel Drosophila SNARE protein 4057 proteins were purified on glutathione beads and cleaved from the GST Fig. 1A), which classify the proteins as Q-SNAREs (Fasshauer moiety by thrombin (Pharmacia protocol; see also Kee and Scheller, et al., 1998). 1996), followed by two 15 minute incubations with 0.3 µM We determined the chromosomal location of SNAP-24 by phenylmethylsulfonyl fluoride. Protein concentrations were estimated screening filters containing high density arrays of genomic P1 by Coomassie Blue staining of protein bands after gel electrophoresis, clones and identified its sequence within P1 #DS04886. with bovine serum albumin as a standard. Formation of SDS-resistant Sequencing of the genomic P1 DNA furthermore revealed that complexes was observed after incubating 2 µM of each recombinant protein in binding buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM the SNAP-24 gene is contained within a single exon. This EDTA or 2 mM CaCl2) overnight at 4°C in a volume of 30 µl. 30 µl contrasts to the complex genomic organization of the SNAP- 2× sample buffer (6% SDS) was added and samples were incubated 25 gene in Drosophila with eight exons distributed over more for 5 minutes either at 37°C or 95°C, resolved by electrophoresis on that 120 kb (Risinger et al., 1997). As the P1 containing SNAP- 12% SDS-polyacrylamide gels and transferred onto nitrocellulose 24 had not been mapped, we used both cDNA and P1-DNA to membranes. Membranes were incubated with anti-syntaxin primary localize the gene by in situ hybridization to polytene antibody (8C3) (Fujita et al., 1982) at 1:200 and anti-mouse secondary chromosomes. SNAP-24 mapped to the right arm of antibody and developed using the enhanced chemiluminescence kit chromosome 3 at map position 85 E10-13 (data not shown). (Amersham). By in situ hybridization it was further determined that the gene was removed by Df (3R)by10 (breakpoints ~85 D8-85 E10-13) but not by Df (3R)GB104 (breakpoints ~85 D8-85 E10). RESULTS Available P-element insertions in the 85E region were screened by genomic Southern blot, but none showed insertion into the Identification of SNAP-24 and DSNAP-29 SNAP-24 gene. To identify additional members of the SNAP-25 SNARE family, we designed degenerate oligonucleotides against highly Temporal and spatial expression pattern of SNAP-24 conserved regions of the and screened poly(A)+ Many of the proteins involved in constitutive vesicle selected RNA by RT-PCR. RNA was isolated from 0-8 hour trafficking, such as syntaxin and synaptobrevin, are found at embryos; this corresponds to a time before synapse the earliest stages in fly development, where they are needed development when SNAP-25 transcripts are not detectable for cellularization of the early zygote. To investigate whether (Risinger et al., 1997), thus increasing the likelihood of SNAP-24 could be important for constitutive membrane fusion identifying a novel homologue. The RT-PCR reaction yielded during cellularization or for the regulated membrane fusion one major band of 200 bp, which was subcloned and sequenced that is found in the nervous system, we first determined the to reveal a novel homologue of SNAP-25. To isolate a full- developmental profile of SNAP-24 transcripts. As predicted length cDNA we screened a cDNA library made from mRNA from its genomic and cDNA structure, a SNAP-24 probe isolated from 0-22 hour embryos (LD library, see Materials and recognized a single transcript of approx. 1.6 kb on a membrane Methods) using the PCR product as a probe and after multiple containing total RNA extracts from the different developmental rounds of screening, isolated a single clone of 1.6 kB. A stages (Fig. 1B). The transcript was highly expressed in complete amino acid sequence was assembled and the embryos at early stages during cellularization and throughout predicted 212 aa protein shows 71% amino acid identity with embryonic life, including the later stages that correlate with Drosophila SNAP-25 (Fig. 1A). Because of the predicted development of the nervous system. Expression levels drop at molecular mass of just under 24 kDa, we named the protein the onset of larval development, but transcript is present at SNAP-24 and deposited the cDNA sequence in GenBank under lower levels in all stages (Fig. 1B). Early embryonic transcript the accession number #AF187106. Similar to SNAP-25, levels were also seen for syntaxin and synaptobrevin (Chin et SNAP-24 contains two aa clusters that score highly on coiled- al., 1993; Parfitt et al., 1995). SNAP-24 transcript was also coil prediction programs. Also conserved are central present in head extracts (data not shown). Because SNAP-24 corresponding to positions 85 and 88 of human SNAP-25, two transcript was detected in 0-2 hour embryos and little or no of the four residues that can be palmitoylated and thus provide zygotic transcription occurs at this time, SNAP-24 mRNA is sites for membrane attachment of SNAP-25 family members likely to be contributed maternally. (Lane and Liu, 1997; Vogel and Roche, 1999). SNAP-24 We generated polyclonal antisera against two clearly is a member of the SNAP-25 family and more distantly nonoverlapping regions of SNAP-24 that have low homology related to human SNAP-29 and to syntaxins. Although SNAP- to SNAP-25 (marked M24 and C24 in Fig. 1A). The specificity 24 is most similar to Drosophila SNAP-25 in sequence it may of antibody binding was tested against 1 µg recombinant be functionally closer to vertebrate SNAP-23, as discussed SNAP-25 or SNAP-24 protein: both antibodies recognize below. SNAP-24 but not SNAP-25 (Fig. 2A). The C24 antibody was Completion of the genome sequencing project (Adams et al., the most robust for immunoblots and immunocytochemistry 2000) confirmed our SNAP-24 sequence and map position and and was used for most of the experiments described here. The also enabled us to identify a more distantly related SNAP-29 M24 antibody, however, served as a confirmation of staining homologue by tblastn searches. patterns because it recognized a distinct region. The C24 The closest Drosophila relative (CG11173) of rat SNAP-29 antibody recognized native protein in head extracts with an maps to 60 A3-5. Although the discovered gene (CG11173) is apparent molecular mass similar to that of the recombinant labeled a putative transporter in the gadfly annotation database, protein. Head extracts of wild-type flies were compared to sequence alignment (Fig. 1A) and analyses of protein structure those of heterozygous flies (Df (3R)by10/+) and, as expected, suggest that CG11173 is indeed the homologue of SNAP-29. SNAP-24 protein levels were reduced in heads that contain Note the conserved in all homologues (asterisks in only one copy of the gene, thus confirming that the antibody 4058 B. A. Niemeyer and T. L. Schwarz

A D24 ------M A A 3 D25 ------M P A 3 H25 ------H23 ------H29 - MSAYPKSYNP- FDDDGEDEG ------A R - P A P W R D A 28 D29 M A H N Y L Q P V H D H F D D V D R F E D V D D D L F L Q N K R T G A A K L P Q Q R S T N P F E M D 50

D24 - - - - - V E N A E P R T E L Q E L Q F K S G Q - - V A D E S L E S T R R M L A L M D E S K E A G I 46 D25 D P S E E V A P Q V P K T E L E E L Q I N A Q G - - V A D E S L E S T R R M L A L C E E S K E A G I 51 H25 - - - - M A E D A D M R N E L E E M Q R R A D Q - - L A D E S L E S T R R M L Q L V E E S K D A G I 44 H23 ------M D N L S S E E I Q Q R A H Q - - I T D E S L E S T R R I L G L A I E S Q D A G I 39 H29 R D L P - D G P D A P A D R Q Q Y L R Q E V L R - - R A E A T A A S T S R S L A L M Y E S E K V G V 75 D29 D D D E E E I T S S P S V A A Q R L A Y A E K R R A I E Q R T L D S T N K S L G L L Y E T Q E V G K 100

D24 R T L V A L D D *Q G E Q L D R I E E G M D R I N A D M R E A E K N L S G M E K C C G - - I C V L P W 94 D25 R T L V A L D D Q G E Q L D R I E E G M D Q I N A D M R E A E K N L S G M E K C C G - - I C V L P C 99 H25 R T L V M L D E Q G E Q L E R I E E G M D Q I N K D M K E A E K N L T D L G K F C G - - L C V C P C 92 H23 K T I T M L D E Q K E Q L N R I E E G L D Q I N K D M R E T E K T L T E L N K C C G - - L C V C P C 87 H29 A S S E E L A R Q R G V L E R T E K M V D K M D Q D L K I S Q K H I N S I K S V F G G L V N Y F K S 125 D29 A T A V E L A K Q R E Q L E K T S H Q L D E I S S T L R F S Q R H L T G L K S V F G G L K N Y L S G 150 M24 D24 K K V N - I K - - D D G E S A W K A N D D G K - - - I V A S Q ------P- Q R V I D E R 127 D25 N K S Q S F K - - E D - D G T W K G N D D G K - - - V V N N Q ------P- Q R V M D D R 132 H25 N K L K - S S - - D A Y K K A W G N N Q D G - - - - V V A S Q ------P- A R V V D E R 124 H23 N R T K N F E S G K A Y K T T W G D G G E N S P C N V V S K Q ------P- G P V T N G Q 126 H29 K P V E T P P E Q N G T L T S Q P N N R L K E A I S T S K E Q E A K Y Q A S H - N - P- L R K L D D T 173 D29 N R D Q - P P T A T G S P T G S Q S S Q E A N - S N I N Q G A C G G A S P S APL S P A E R Y D N H 198

D24 E R G G M G A P P Q S G Y V A R I T N - - - D A R E D E M D E N L G Q V N S M L G N L R N M A L D M 174 D25 N - - G M M A - - Q A G Y I G R I T N - - - D A R E D E M E E N M G Q V N T M I G N L R N M A L D M 175 H25 E - - Q M A I - - S G G F I R R V T N - - - D A R E N E M D E N L E Q V S G I I G N L R H M A L D M 167 H23 L Q Q P T T G A V S G G Y I K R I T N - - - D A R E D E M E E N L T Q V G S I L G N L K D M A L N I 173 H29 D P V P R G A G S A M S T D A Y P K N P H L R A Y H Q K I D S N L D E L S M G L G R L K D I A L G M 223 D29 P V S Q L R G D P S S T Y Q P Q R Q A A - - N P F Q A Q I D S N L E E M C S N L S V L K M L A T D L 246 C24 D24 G S E L E N *Q N K Q V D R I N A K G D A N N I R M D G V N K R A N N L L K S 212 D25 G S E L E N Q N R Q I D R I N R K G E S N E A R I A V A N Q R A H Q L L K 212 H25 G N E I D T Q N R Q I D R I M E K A D S N K T R I D E A N Q R A T K M L G S G 206 H23 G N E I D A Q N P Q I K R I T D K A D T N R D R I D I A N A R A K K L I D S 211 H29 Q T E I E E Q D D I L D R L T T K V D K L D V N I K S T E R K V R Q L 258 D29 G G E I E S Q N E L L D N M N Y K I E D V D L K I H K Q N K D M S K L L K K 284 BoNT/E BoNT/A

Fig. 1. (A) Sequence of SNAP-24. Alignment of the Drosophila SNAP-24 (D24) sequence with that of Drosophila SNAP-25 (D25) human SNAP-25 (H25), human SNAP-23 (H23) and the putative Drosophila SNAP-29 homologue (D29) (GenBankTM accession numbers P36975, NP_003072, AAC50537, CG11173). Identical residues in all six proteins are shaded dark gray, identical residues in four are shaded light gray. Sequences are numbered on the right. Sequences used in the generation of peptide antibodies are overlined (M24 and C24). Toxin cleavage sites are marked by arrows and asterisks indicate the glutamine residues that define these proteins as Q-SNAREs. (B) Developmental transcript analysis of SNAP-24. 20 µg of total RNA from the indicated developmental stages were size-fractionated on a 0.8% agarose/formaldehyde gel, transferred onto nitrocellulose membrane and hybridized at high stringency with first a SNAP-24 specific probe and subsequently a probe against ribosomal protein 49 (rp49) as control. The SNAP-24 transcript is about 1.6 kb in size and is present in all developmental stages investigated, albeit more concentrated during embryonic development. Note the presence of maternally contributed RNA in 0-2 hour embryos. recognizes the correct protein. To control for sufficient protein confocal sections of whole-mount third instar larval brain loading the extracts were simultaneously probed for the lobes, Fig. 3F,G shows a lower magnification view of whole- presence of syntaxin using a monoclonal anti-syntaxin mount brain and ventral nerve ganglia. SNAP-24, as visualized antibody (Fig. 2B). Similar results were obtained in protein with the C24 antibody, was seen in a punctate pattern at low extracts from larval brains, salivary glands, adult testes, ovaries levels throughout the brain and ventral nerve ganglia; the and bodies (data not shown). puncta were seen at different depths of focus. The punctate To investigate SNAP-24 protein expression in the nervous pattern was abolished when the antibody was preabsorbed with system, we looked at the distribution of both SNAP-25 and peptide (Fig. 3C). A similar punctate staining pattern was also SNAP-24 in the brain and ventral ganglia. Fig. 3A,B shows seen with the M24 antibody (data not shown). The puncta SNAP-24, a novel Drosophila SNARE protein 4059

crossreactivity of the syntaxin antibody as seen on western blots of recombinant proteins (see below) does not interfere significantly at the immunohistochemical level. We have also stained similar stage brains with an antibody against neuronal synaptobrevin (n-syb), which is found on vesicles. As expected, n-syb was highly expressed in the neuropil and synaptic regions but, interestingly, also in some puncta outside the neuropil (data not shown). By analogy to the salviary glands described below, it appears likely that the puncta resemble nonsynaptic exocytotic vesicles destined for the plasma membrane. SNAP-24 can form SDS-resistant complexes but cannot be cleaved by Botulinum E Fig. 2. Specificity of SNAP-24 antibodies. (A) Specificity of SNAP- Because SNAP-24 appeared to be involved in trafficking 24 antibodies was tested by immunoblot analysis of 1 µg each of outside the synapse, we asked what its partners might be for recombinant SNAP-24 and SNAP-25 protein. (B) Native SNAP-24 forming core complexes and whether there would be selectivity protein is present in extracts of adult heads (3.13 heads/lane) and between SNAP-25 and SNAP-24 in recognizing different protein levels are reduced in Df(3R)by10/+ flies, which contain only isoforms of the vesicular SNAREs n-syb and syb. We one copy of the gene (Def/+). Blots were simultaneously probed for incubated equimolar amounts of purified recombinant SNARE the presence of syntaxin to control for protein loading. proteins and monitored the formation of SDS-resistant high molecular mass complexes (Sollner et al., 1993). The complexes were then size-fractionated and visualized with an appear to be within cells and, rather than being restricted to the anti-syntaxin antibody (Fig. 4A). Two major complexes synaptic regions that form the central core of the brain lobes migrating at 55-60 kDa and at approx. 95 kDa on SDS gels and ganglia, are scattered throughout the tissue. In contrast to were formed in the presence of two t-SNAREs and one v- SNAP-24’s non-synaptic label, SNAP-25 was expressed SNARE. Boiling the samples dissolved both complexes and no almost exclusively in the neuropil and synaptic regions (Fig. SDS-resistant complex was formed if only two binding 3B,G). partners were present. The small contaminating band at approx. To confirm SNAP-24 staining and to circumvent potential 60 kDa that is resistant to boiling was due to a small amount accessibility problems, we double-labeled cryostat sections of of syntaxin still linked to the GST protein, which may also have larval brains with the C24 antibody and with a monoclonal contributed to the appearance of two complexes. Note that anti-syntaxin antibody (Fujita et al., 1982). Fig. 3D shows that, although a monoclonal antisyntaxin antibody was used, the while puncta of SNAP-24 could be seen in the neuronal cell antibody crossreacts with recombinant SNAP-24 and SNAP- body region (CB), syntaxin (Fig. 3E) was expressed in the 25 protein, suggesting either that the antigenic epitope is central neuropil and synaptic region of the brain (NP). The conserved between the three proteins or that the ascitis

Fig. 3. SNAP-24 and SNAP-25 expression in the nervous system. Indirect immunofluorescence staining of larval brain lobes [B] and ventral- nerve ganglia [VNG] in whole-mount preparations (A-C,F,G) or cryostat sections (D,E). (A,F) Punctate SNAP- 24 immunoreactivity visualized with C24 antibody in a confocal image of third instar brain and ventral nerve ganglion (F) and of a single brain lobe (A). (C) A confocal image of a brain lobe treated with the same concentration of C24 antibody with the addition of blocking peptide. SNAP-25 staining, unlike SNAP-24, is seen concentrated in the synapse-rich neuropil of both the brain lobes (B) and the ventral nerve ganglion (G). (D,E) Double-labeled cryostat sections of larval brain show that SNAP-24 puncta (D) are concentrated in the cell body region [CB] while syntaxin (E) is found primarily in regions rich in axons and synapses [NP]. Scale bars, 20 µm (F,G); 10 µm (A-E). 4060 B. A. Niemeyer and T. L. Schwarz

Fig. 4. SNAP-24 can form SDS- resistant complexes but cannot be cleaved by Botulinum neurotoxin E. (A) SNAP-24 and SNAP-25 can form SDS-resistant complexes with syntaxin 1A and neuronal synaptobrevin (N- Syb) or with syntaxin and synaptobrevin (Syb). Western blot showing complex formation visualized with anti-syntaxin (8C3) antibody. The faint band at approx. 55 kDa that is not removed by boiling is due to a small amount of uncleaved GST-syntaxin. Most of the 55 kDa complex and all of the complex between 77 and 103 kDa are dependent on the presence of three different SNARE protein partners, though no preference for any given t- or v-SNARE can be detected. Note that the anti-syntaxin antibody crossreacts with recombinant SNAP- 24 and SNAP-25. (B) Cleavage by Botulinum neurotoxin E: Coomassie Blue-stained blot of approx. 1.5 µg recombinant protein treated with increasing concentrations of recombinant BoNT/E light chain for the times indicated. Complete cleavage of mouse SNAP-25 was obtained with 100 nM toxin treatment for 6 minutes. Note that both Drosophila SNAP-24 (Dro 24) and Drosophila SNAP-25 (Dro 25) are not efficiently cleaved: at the highest toxin concentration tested most of the recombinant protein remained in the uncleaved form. The band of approx. 25 kDa at very high toxin concentration is a contaminant of the toxin preparation. supernatant did not derive from pure clonal cells. No specificity Torpedo marmorata SNAP-25 (Washbourne et al., 1997). was seen in the ability of these components to form complexes. While mouse SNAP-25 was completely cleaved by a 6-minute The t-SNAREs SNAP-24 and syntaxin bound either n-syb or treatment with 100 nM BoNT/E light chain, neither Drosophila syb tightly enough to be stable in SDS. Similarly, SNAP-25 SNAP-24 nor Drosophila SNAP-25 was cleaved (Fig. 4B). and syntaxin did not show a preference for either syb or n-syb Even at a 26-fold higher toxin concentration, no significant and formed a complex with both. We also tested complex reduction of uncleaved protein was seen (see also Washbourne formation in the presence of 2 mM and again no et al., 1997, for Drosophila SNAP-25). Therefore, strategies significant differences were found in the ability of either based on in vivo cleavage of the Drosophila t-SNAREs SNAP- SNAP-24 or SNAP-25 to form complexes (data not shown). 24 and SNAP-25 are not likely to succeed. Because cleavage by BoNT/A or BoNT/E may provide a tool to study the function of SNAP-24 (see Chen et al., 1999b), SNAP-24 is highly expressed in salivary gland cells we investigated the susceptibility of recombinant protein Compared to the relatively weak staining of SNAP-24 in the towards toxin cleavage. The SNAP-24 sequence has a nervous system, much stronger immunoreactivity was found in conserved BoNT/E cleavage site (Fig. 1A, arrow), but contains whole mounts and 12 µm thin cryostat sections of third instar a glutamine to lysine change at the BoNT/A cleavage site, larval salivary glands (Fig. 5A). Confocal images were similar to the amino acid change that prevents cleavage of acquired at approx. 75% of the photomultiplier sensitivity used

Fig. 5. SNAP-24 is highly expressed in salivary gland cells. Confocal images of whole-mount third instar salivary glands. (A) Secretory cells stained with C24 anti-SNAP-24 antibody. (B) Syntaxin 1A expression is concentrated at the apical cell surfaces lining the lumen of the gland. (C) Two salivary glands stained with peptide-blocked C24 antibody. Laser intensity and PMT sensitivity same as in A. Scale bars, 20 µm. SNAP-24, a novel Drosophila SNARE protein 4061

Fig. 6. SNAP-24 redistributes during exocytotic events at early pupariation. (A,D,G) Low magnification images of whole-mount salivary glands at third instar (A), at onset of glue secretion (D) and towards the end of glue secretion (G). Note the depletion of vesicles from the basolateral surfaces in D and the vast expansion of luminal surfaces in G. Higher magnification views (B,C) demonstrate that SNAP-24 is expressed on membranes of large secretory granules during third instar larval development. These vesicles fuse with one another at the onset of pupation (E,F). The arrows in F point to examples of multigranular fusion complexes. As granules fuse at the apical surface, the lumen of the gland expands and begins to invade basolateral regions of the cells (H). The arrow points to a multigranular complex fusing with the apical membrane. (I) Salivary gland cell volume is now greatly reduced with few SNAP-24 positive vesicles remaining and most immunoreactivity transferred to the enlarged apical surface (thick arrow), which is now within 10 µm of the exterior of the gland (thin arrow). Scale bars, 20 µm (A,D,G); 10 µm (B,E); 5 µm (H,C,F,I). for imaging the central nervous system (Fig. 3). SNAP-24 secrete glue proteins are full of SNAP-24 coated granules, we staining had an uneven appearance over the surface of whole- hypothesized that SNAP-24 may play a role in glue protein mount glands and could be blocked by preabsorption with the secretion. We looked in more detail at salivary glands dissected C24 peptide (Fig. 5C). The staining appears to correspond from third instar larvae and larvae at the larval/pupal transition to the surface of secretory granules that fill these cells period, where glue protein is secreted. Whole glands were (see below). In cryostat-sectioned material, SNAP-24 was dissected from crawling third instars, transitional third instars distributed on granules throughout each secretory cell. Similar that had stopped moving and shortened, and from white SNAP-24 staining was also observed using the M24 antibody prepupae, and their salivary glands were stained with anti- and this signal could also be blocked by preabsorption with the SNAP-24 antibody (Fig. 6). Whereas SNAP-24 had a granular M24 peptide (data not shown). In contrast, syntaxin was distribution throughout the cells of a late third instar gland (Fig. concentrated on the apical membranes of the gland cells and 6A), glands from transitional third instar larvae had a much more thereby outlined the salivary gland lumen (Fig. 5B). The faint irregular granular stain with areas that appear depleted of signal signal on the basolateral surfaces may be due to much lower (Fig. 6D). A short time later, the granular appearance was almost levels of syntaxin or to a low level of crossreactivity. The strong gone and immunoreactivity was evenly dispersed (Fig. 6G). staining on apical membranes was also seen in cryostat High magnification images of the gland cells revealed that in late sections and thus was not an artifact of poor penetration by the third instar cells SNAP-24 was expressed on membranes of large antibody. Smaller cells that line the shaft of the gland (imaginal (3.5 µm average diameter) tightly packed granules (Fig. 6B,C). adult salivary gland cells; Ross, 1939) were not labeled No significant label could be found on basolateral plasma with SNAP-24 antibodies. We have also tested SNAP-25 membranes of these cells and very little label on the apical immunoreactivity in salivary glands and find little signal within membrane surfaces (data not shown). At the onset of glue third instar gland cells, though positive signal was seen in the secretion, these granules appeared to fuse with one another and imaginal adult gland cells lining the shaft of the salivary gland form multigranular fusion complexes (Fig. 6E,F). It does not (data not shown). appear that the granules were merely aggregating because, when imaged at the right depth of section, SNAP-24 labeled the SNAP-24 redistributes during exocytotic events at outside of the larger fusion complexes and did not divide the early pupariation darker contiguous lumens (see arrows in Fig. 6F). Probably Salivary glands in Drosophila synthesize and store concomitant with granule to granule fusion, fusion of granules mucopolysaccarides (glue proteins) in their cells during the and multigranular complexes with the apical plasma membrane third instar (Berendes, 1965; Poels, 1972). These glue proteins surface could be seen (Fig. 6H) and the surface area of the lumen are secreted into the gland lumen at the onset of puparium expanded (Fig. 6G,I). As the granules fuse with the apical formation and fix the puparium to a substrate (Beckendorf and membrane, the luminal surface invades more basolateral regions Kafatos, 1976; Fraenkel and Brookes, 1953). of the cells and more SNAP-24 appears at the apical plasma Given our observation that salivary gland cells destined to membrane. Towards the end of glue secretion, the luminal 4062 B. A. Niemeyer and T. L. Schwarz

A In the present study we have examined the potential of distinct isoforms of SNAP-25 for selecting among the varieties 40 of trafficking to the plasma membrane. Genetic data suggest that distinct v-SNAREs are involved in the traffic of these 30

vesicles populations of vesicles: syb, which is required for cell viability

of 20 (S. Battacharya and T. L. Schwarz, unpublished data), and n- syb, which is specifically required for the rapid, Ca2+-triggered 10 release of at nerve terminals (Deitcher et al.,

Number 1998; Yoshihara et al., 1999). Because both processes depend 0 on a single syntaxin gene (Burgess et al., 1997; Schulze et al., 0502525 75 100 125 150 1995), any specificity in SNARE-pairing must lie in the Area (µ m2 ) B selection of isoforms of SNAP-25. Only a synaptic isoform had been previously identified in Drosophila. Therefore, we 1.2 screened for an additional fly gene and uncovered a homologue 1.0 we have called SNAP-24. The distribution of SNAP-24 in the 0.8 organism and within the nervous system indicated that it is 0.6 likely to be a functional partner for syb; SNAP-24 is present Frequency 0.4 at all developmental stages, including those before neuronal 0.2 differentiation when syb is the dominant v-SNARE isoform. 0.0 Norm. Within neurons, SNAP-25 is concentrated (like n-syb) at 0 20 40 60 80 100 120 140 synapses, while SNAP-24 is predominantly in the cell body. Area (µ m2) However, despite the suggestive differential distribution of these isoforms, we could not detect any differences in the Fig. 7. Quantification of vesicle size. (A) Histogram showing the distribution of cross-sectional areas of third instar granules (open biochemical interactions of SNAP-24 and SNAP-25 that could hatched bars) versus multigranular complexes from transitional provide a substrate for selective targeting or fusion: both larvae (solid bars). Areas of granules were determined by measuring isoforms failed to discriminate between syb and n-syb proteins their diameter; areas of multigranular complexes were determined by in vitro and formed stable, SDS-resistant complexes with both. image analysis of their outlined area. (B) Cumulative frequency The biochemical similarities of SNAP-24 and SNAP-25 distribution of the data in A for third instar granules (circles) described above do not suggest a functional purpose for their compared to multigranular complexes from transitional larvae evolutionary divergence, though they must contain targeting (triangles). The data sets were significantly different (P<0.01) by sequences that determine their differential distribution within Kolmogorov-Smirnov analysis. cells. A clue for further biochemical studies may lie in the existence of two NSF genes in Drosophila (Boulianne and surface is in close proximity to the basal membrane and only a Trimble, 1995; Ordway et al., 1994; Pallanck et al., 1995b). few granules remain in the greatly shrunken of the NSF1 (comatose) is necessary for synaptic transmission at gland cells (Fig. 6I). To quantitate the events we interpret as some neuromuscular junctions (Kawasaki et al., 1998; Pallanck compound exocytosis, we measured granule sizes before and et al., 1995a; Siddiqi and Benzer, 1976). NSF2, however, has during glue secretion. A striking increase in the number of a distribution that is somewhat reminiscent of SNAP-24; NSF2 granules with large areas was seen between third instar and is expressed in the nervous system during embryogenesis and transitional SNAP-24 positive granules (Fig. 7A). The two in several imaginal discs and is particularly abundant in the populations of granules were significantly different, as also larval salivary glands (Boulianne and Trimble, 1995). Perhaps shown in a cumulative frequency distribution of the two samples NSF2 has specialized to interact with SNAP-24 containing (Fig. 7B) (P<0.01; Kolmogorov-Smirnov analyses). core complexes. The Drosophila SNAP-24 gene we describe above shows greater sequence identity with Drosophila SNAP-25 than with DISCUSSION any of the mammalian isoforms, and is slightly closer to mammalian SNAP-25 than to mammalian SNAP-23. Yet, it In neurons, multiple classes of vesicular transport to the plasma seems possible that this gene is a functional homologue of the membrane occur. Synaptic vesicles are specifically targeted to mammalian SNAP-23 gene. Mammalian SNAP-23 is and fuse with presynaptic membranes. Other ubiquitously expressed, and can bind to multiple syntaxins and vesicles destined for the plasma membrane, including for /VAMPs (Ravichandran et al., 1996). Like example those bearing glutamate receptors, are trafficked to SNAP-25, it can be localized to the plasma membrane (Araki postsynaptic sites (Shi et al., 1999; Stowell and Craig, 1999). et al., 1997; Chen et al., 1997) with a membrane anchor The mechanism by which this distinction in targeting is provided by of central cysteines (Vogel and accomplished remains unclear. The SNARE hypothesis Roche, 1999). Functions for SNAP-23 include insulin- proposed a central role for the cognate pairing of v- and t- dependent glucose uptake in adipocytes (Foster et al., 1999), SNAREs in the specification of transport to appropriate target apical trafficking in kidney cells (Lafont et al., 1999; Leung et membranes (Rothman and Wieland, 1996; Scales et al., 2000; al., 1998; Low et al., 1998) and platelet alpha-granule secretion Sollner et al., 1993), but this hypothesis has been open to (Flaumenhaft et al., 1999). In mammalian brain tissues, SNAP- question (Broadie et al., 1995; Deitcher et al., 1998; Fasshauer 23 has been found in glia (Hepp et al., 1999) and in the cell et al., 1999; Hunt et al., 1994; Yang et al., 1999). bodies of neurons (Chen et al., 1999a). This diverse SNAP-24, a novel Drosophila SNARE protein 4063 distribution for nonsynaptic membrane trafficking may plasma membrane but also by vesicle-to-vesicle fusions, is a correlate with the diverse tissue distribution of Drosophila widespread phenomenon that occurs in many secretory cells SNAP-24 and suggests a similar function. Interestingly, upon including mast cells (Alvarez de Toledo and Fernandez, 1990; stimulation of secretion in mast cells, SNAP-23 translocates Chandler and Heuser, 1980; Rohlich et al., 1971) and from the plasma membrane to the secretory granules pancreatic cells (Hansen et al., 1999). The presence of SNAP- (Guo et al., 1998) and is involved in regulating compound 24, syntaxin1 and NSF2 in the salivary glands of Drosophila exocytosis. SNAP-24 in the salivary gland appears to mediate cells should provide a starting point for a genetic analysis of a similar case of compound exocytosis. Unlike SNAP-23, compound exocytosis. Such studies may further elucidate the however, there is no indication that SNAP-24 originates on the manner in which the distribution of the fusion machinery is plasma membrane and translocates to the granules prior to altered to accomplish this specialized form of secretion. fusion. On the contrary, we observe the majority of the SNAP- 24 immunoreactivity on the secretory granules and, as those We especially thank Dr David Deitcher for allowing us to use his granules merge with the plasma membrane, it becomes specific SNAP-25 antibody and Drs Suzie Scales and Richard Scheller abundant on the apical membrane. for their generous contribution of mammalian SNAP-25 and BoNT/E Vertebrates also contain SNAP-29 (Steegmaier et al., 1998), light chain. Thanks also to Mala Murthy and Drs Ege Kavalali, Erika Piedras and Steve Stowers for helpful suggestions. a more distant relative of SNAP-25, which lacks the cysteines that provide the membrane anchor. The Drosophila genome, however, contains a gene distinct from the one we report, REFERENCES which is more similar to SNAP-29 than to the SNAP-23/25 branch of the tree. SNAP-24 is therefore unlikely to be the Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., functional homolog of SNAP-29. Amanatides, P. 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