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SNAP-24, a Novel Drosophila SNARE Protein 4057 Proteins Were Purified on Glutathione Beads and Cleaved from the GST Fig

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SNAP-24, a Novel Drosophila SNARE Protein 4057 Proteins Were Purified on Glutathione Beads and Cleaved from the GST Fig Journal of Cell Science 113, 4055-4064 (2000) 4055 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1894 SNAP-24, a Drosophila 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 protein) syntaxin and both synaptic and non-synaptic v-SNAREs. attachment protein receptor) proteins 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 synapses and additional t-SNAREs must exist that mediate containing granules fuse with one another and the apical both non-synaptic fusion in neurons and constitutive and membrane, suggesting that glue secretion utilizes regulated fusion in other cells. Here we report the compound exocytosis 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 synaptobrevin/VAMP) and recently been demonstrated in a reconstituted PC12 secretion corresponding partners such as syntaxins 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 neurotoxins Botulinum toxin 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 neurotransmission (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 tetanus toxin or disruption of the n-syb gene prevents action- to an appropriate membrane (Rothman and Wieland, 1996; potential-evoked release at the neuromuscular junction, 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 homology to SNAP-25. transport vesicle to plasma membrane fusion outside the Antibodies synapse, 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- neuron. 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 cysteine 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 enzyme 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).
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