Characterization and gene cloning of Drosophila syntaxin 1 (Dsynt1) the fruit fly homologue of rat syntaxin 1

MOLECULAR BRAIN RESEARCH

IIPT!~!iI' Ill ELSEVIER

Molecular Brain Research 29 (1995) 245-252

Research report

Characterization and gene cloning of Drosophila syntaxin 1 (Dsyntl)" the fruit fly hornologue of rat syntaxin 1 Jose R. Cerezo a, F e r n a n d o Jim6nez b, F e r n a n d o Moya ~'* Departamento de Gengtica y Microbiologfa and lnstituto de Neurociencias, Universidad de Alicante, 03080, San Juan, Alicante, Spain b Centro de Biologfa Molecular Severo Ochoa, Unieersidad Aut6noma-CSIC, 28049, Madrid, Spain Accepted 25 October 1994

Abstract A monoclonal antibody, mAb 44D5, has been used to identify and clone Drosophila syntaxin 1 (Dsyntl), an homologue of rat syntaxin 1. The deduced amino acid sequence of the Dsyntl cDNA cloned is highly homologous to rat syntaxin 1A. Dsyntl contains 291 amino acid residues and like other members of the syntaxin family is an integral membrane protein, with a transmembrane region at its carboxy-terminus and several regions of the molecule predicted to be in a coiled-coil conformation. The protein is specific to the nervous system and localized in synaptic areas of both central nervous system (CNS) and neuromuscular junction. The same antibody used to clone Dsyntl cDNA stains synaptic areas in rat cerebellum and a neuroespecific antigen in rat and human tissues with identical relative mobility to rat syntaxin 1.

Keywords." Syntaxin; Drosophila; Dsyntl; Synaptic transmission ; Synaptic proteins; Neurotransmitter release

I. Introduction Synaptic function involves the assembly of synaptic vesicles on a specific region of the presynaptic m e m brane known as the active zone, the fusion of synaptic vesicles to the plasma m e m b r a n e in response to a local increase in Ca 2+ concentration mediated through voltage-dependent calcium channels [16,27], and the subsequent release of their content to the synaptic cleft. These processes, docking and fusion of synaptic vesicles and the release of neurotransmitter, require the temporal and spatial regulation of diverse interactions between proteins at the synaptic vesicles and the presynaptic membrane. A n u m b e r of proteins that could participate in these processes have been identified (for a review see ref. [11]). One of these proteins, syntaxin 1, is an integral m e m b r a n e protein thought to participate in the docking a n d / o r fusion of vesicles and that is present in at least two isoforms (1A and 1B) in the rat nervous system [2]. Syntaxin 1, shares sequence and structural homology with other rat proteins, syntaxins 2-5, each of them expressed with a distinct tissue-specific distri-

* Corresponding author. Fax: (34) 6 5941787 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 2 5 4 - 1

bution. The proteins of the rat syntaxin family show also an analogy with proteins from other organisms, including some yeast proteins, with the general function of directing intracellular vesicles traffic to the plasma membrane, which have been described with the generic name of SNAP receptors or S N A R E s [25,26]. Therefore, syntaxins 1A and 1B seem to be the neuronal representatives of a large family of proteins involved in the targeting of secretory vesicles through the cell. The functional role of a specific neuronal protein of this class would be linked to the necessary spatial and temporal coupling of neurotransmitter release to the physiological events that take place at the synaptic terminal. In fact, evidence suggests that syntaxin 1A and 1B could serve as a link between the N-type Ca-channels involved in neurotransmitter release and synaptic vesicles, via some of the synaptic vesicle proteins: synaptotagmin (p65) [1,14,29] and vesicle-associated m e m b r a n e proteins (VAMPs) 1 and 2 [4]. Interactions of all these proteins could form a secretory complex including the a-latrotoxin receptor (neurexin) [19] and a 67 K protein homologue of the unc-18 gene of C. Elegans [9,21]. In this article we report on the characterization and gene cloning of Drosophila syntaxin 1 (Dsyntl). The deduced amino acid sequence of 291 residues is highly

246

J.R. Cerezo et aL / Molecular Brain Research 29 (1995) 245-252

homologous to rat syntaxin 1. A monoclonal antibody (mAb 44D5) used for expression cloning of Dsyntl cDNA, recognizes synaptic areas in Drosophila CNS and synaptic boutons at the larval neuromuscular junction, confirming the synaptic location of the protein. Moreover, mAb 44D5 recognizes a vertebrate antigen located at synaptic areas, similar to rat syntaxin 1 in its apparent molecular weight (about 37 kDa) and tissue distribution.

2. Materials and methods

2.1. Monoclonal antibody production Hybridome cell lines were derived by standard methods from a mouse immunized with homogenates of CNS and imaginal discs dissected from third instar Drosophila larvae. Supernatants from the hybridoma cultures were screened by indirect immunofluorescence on third instar larvae cryostat sections. Ascites fluid, purified by ammonium sulfate precipitation or supernatant from hybridoma cultures were used as a source of monoclonal antibodies for further experiments.

2.2. Immunocytochemistry Whole mount embryos were stained as described [18]. Adult heads were fixed overnight in 4% formaldehyde in PBS at 4°C. Cryostat sections of 12 /xm thickness were treated with mAb 44D5 in PBS, 0.1% Tween 20, 0.1% bovine serum albumin (BSA) for 30 min at room temperature. After washing, the sections were incubated with biotinylated secondary antibody and then with the Elite kit (Vector Labs). Horseradish peroxidase (HRP) was developed with diaminobenzidine (DAB). Larval muscles were stained as adult heads following dissection of third instar larvae in Bouin's fixative. Rats of different postnatal (P) ages (P4, P9 and P15) were ether anesthetized and perfused for 20 min with 4% paraformaldehyde and 0.1 M sucrose in 0.1 M phosphate, pH 7.4, at 4°C. Cryostat sections of 25 ~m thickness from cerebella were mounted on gelatinized slides and probed with mAb 44D5 in 0.3% Triton X-100 and 3% fetal calf serum (FCS) for 24 h at 4°C. After washing, the sections were incubated with a HRP-conjugated goat anti-mouse IgG (Nordic Laboratories), and developed with DAB.

reals and kept in the same buffer at -20°C. Pieces from human cerebella were collected from autopsies at Hospital Virgen de los Lirios (Alcoi) and stored at - 2 0 ° C until use. Samples from different sources were homogenized in 50 mM Tris-HC1, pH 7.4, 140 mM NaCI (TBS) containing 1 mM PMSF and 0.5% Triton X-100, sonicated for 1 min and centrifuged at 10.000 x g for 10 min. The pellets were discarded. Total protein was estimated by the method of Lowry et al. [17] and samples diluted to their final concentration; 25 ~zg of protein from each homogenate were electrophoresed on SDS-polyacrilamide gels [12]. Transfer to a nitrocellulose filter was performed according to Towbin et al. [28] in the presence of 0.01% SDS. The filter was preincubated for 30 min in TBS containing 5% goat serum and incubated overnight at room temperature with mAb 44D5 supernatant in 3% FCS and 0.05% Tween 20. The filter was washed in TBS with 0.05% Tween 20 (TBST) and incubated with an alkaline-phosphatase (AP)-labeled goat anti-mouse IgG (Tago). AP was developed by standard procedures.

2.4. Isolation of cDNA Monoclonal antibody 44D5 was used for expression cloning from a 0-20 h embryonic Agtll cDNA library, prepared by random priming (a gift from B. Hovemann). The library was screened essentially as described by Huynh et al. [10], using an AP-labeled secondary antibody. A single clone (D1) was isolated.

2.5. DNA sequencing Eco R1 fragments (670 bp and 430 bp, fragment I and II, respectively) from clone D1 were subcloned using the Bluescript plasmid system for sequencing. DNA sequencing was performed by the dideoxy method [23] using 35S-labeled d(thio)ATP and Sequenase (Stratagene). The sequence was determined for both DNA strands. Sequences were constructed and analyzed with the help of the PCGENE software package.

2. 6. Expression of h gtl 1 recombinant protein Recombinant protein produced by D1 clone was prepared from infected Y1089 lysogens induced with isopropyl/3-D-thiogalactopyranoside (IPTG) 10 mM for 1 h as described [24].

3. Results

2.3. Western blotting 3.1. Selection of mAb 44D5 Third instar larval CNS were dissected in ice-cold Tris-HC1, pH 7.4, 100 mM NaCI and 1 mM PMSF. Rat cerebella were dissected from ether-anesthetized ani-

A monoclonal antibody, mAb 44D5, was isolated in a screening of neurospecific antigens expressed in lar-

J.R. Cerezo et al. / Molecular Brain Research 29 (1995) 245-252

val CNS. The antigen recognized by mAb 44D5 is distributed throughout the nervous system and exclusively located in the neuropil, where synapses are present, not labeling the neuronal cell bodies of the cell cortex. Western blots of larval CNS homogenates showed that mAb 44D5 recognizes a protein of approximately 37 kDa (Fig. 1, lane 3). Proteins of similar relative mobility are recognized by mAb 44D5 in rat and human CNS homogenates (Fig. 1, lanes 2 and 1, respectively). The rat antigen recognized by mAb 44D5 is not present in other tissues than CNS (data not shown). Therefore, the antigen recognized by mAb 44D5 in vertebrates is similar in size and tissue distribution to rat syntaxin 1. Based on these data and the evidence shown below (see sequence analysis) we postulate that the protein recognized by mAb 44D5 is the Drosophila homologue of rat syntaxin 1 and we will refer to it as Dsyntl.

3.2. Cloning of Drosophila syntaxin 1 (Dsyntl) cDNA The same antibody (mAb 44D5) was used to screen an embryonic cDNA expression library. A single clone (D1), expressing immunoreactivity and containing an insert of approximately 1100 bp, was isolated. Expression in E. Coli produced a recombinant protein of about 34 kDa which is recognized by mAb 44D5 (Fig. 2, lane 2). This result indicates that expression of the recombinant protein coded by D1 is under the control of the [3-gal promoter but it is not expressed as a /3-galactosidase fusion protein. Therefore, D1 seems to encode and express a protein of about 34 kDa as a lacZ-dependent operon fusion. This recombinant protein, slightly smaller than the antigen recognized in

kl)d

45,

31,

21,

I

2

3

Fig. 1. Western blot analysis with m A b 44D5 of neural tissue, Proteins from different homogenates were run in a 12% polyacrylamide Laemmli gel, transferred to nitrocellulose and treated with m A b 44D5 as described in Materials and methods. Samples were homogenates of h u m a n adult cerebellum from autopsy (lane 1), cerebellum from a 9 day postnatal rat (lane 2) and third instar Drosophila larvae CNS (lane 3). Size was estimated using standards of known molecular weight.

247

kOa

66,~

45*

!

2

Fig. 2. Western blot analysis of the recombinant protein produced by clone D1. Proteins were run in a 7.5% polyacrylamide Laemmli gel and treated as in Fig. 1. Lane 1: homogenates of 9 day postnatal rat cerebellum (same as in Fig. 1). Lane 2: homogenate of Y1089 lysogens, after induction with IPTG (10 raM) for 1 h at 37°C. Notice the presence in lane 2 of a band reacting with mAb 44D5 and running slightly faster (about 34 kDa) than the protein present in rat neural tissue (lane II.

Drosophila, could be a truncated form of Dsyntl at either end, or simply correspond to the full length Dsyntl without postranslational modifications. Sequence analysis of the DNA encoded by D1 (see bellow) revealed that it contains the full length coding region of the putative Dsyntl cDNA, including a few nucleotides upstream and downstream of the translated sequence.

3.3. Sequence analysis of Dsynt l DNA We subcloned the insert in clone D1 into the pBlueScript system in order to sequence it. EcoRI treatment of D1 produced two fragments of approximately 670 bp (fragment I) and 430 bp (fragment II) that were subcloned and sequenced separately. The sequences of both fragments were aligned overlapping the EcoRl sites in order to give the longest open reading frame (ORF) possible which would code for a protein of 291 amino acid residues, MW of 33649. Therefore, the total length of the insert in the original clone would correspond to a 1066 bp insert containing an O R F that would code a protein of about 34 kDa, a size equivalent to that of the protein expressed by D1 (Fig. 2, lane 2). The 873 bp O R F is flanked by 69 bp 5' of the translation initiation site, and 123 bp 3' of the stop codon at position 943. The 5' segment, upstream of the triplet coding the initial methionine, includes stop codons in each reading frame, a fact that could explain the expression of Dsyntl in clone D1 as a non-fusion protein under the control of the 13-gal promoter. We concluded that this alignment reflects the order and orientation of fragments I and II in clone D1. The 3' end does not include a polyA segment and therefore

J.R. Cerezo et al. /Molecular Brain Research 29 (1995) 245-252

248

Table 1 Homology of Drosophila syntaxin 1 with rat syntaxins

Identity Similarity Total

synla

synlb

syn2a

syn3

syn4

syn5

68.4 13.2 81.6

68.06 11.81 79.9

56.90 15.86 72.8

55.02 16.61 71.6

39.18 16.15 55.3

20.27 13.75 34.0

calculated identity and similarity percentages with different members of the rat syntaxin family. Drosophila syntaxin 1 is most closely related to rat syntaxin 1A (81.6% homology) and rat syntaxin 1B (79.9% homology) showing a more distant relationship to the rest of rat syntaxins, as it happens with the two isoforms of rat syntaxin 1. This homology and the neurospecificity of the protein recognized by mAb 44D5, both in Drosophila and vertebrate CNS, suggest that the protein coded by D1 is the Drosophila homologue of the neurospecific rat syntaxin 1, either 1A or lB. Dsyntl structure contains many features characteristic of the syntaxin family. The C-terminal transmembrane region, common to all members of the rat syntaxin family, is present in Dsyntl although the homology between the proteins in this region and their N-terminal end is lower than in the rest of the molecule.

Percentage of total homology for each protein is the addition of identity and similarity values as calculated with the Dayhoff MDM-78 matrix with an open gap cost of 200 and a unit gap cost of 100 (PALIGN program in PCGENE).

D1 does not seem to correspond to the full length mature mRNA for Dsyntl. The deduced amino acid sequence of 291 residues (Fig. 3) shows a strong homology both in its primary and secondary structure with the described sequences for rat syntaxins 1A and 1B [2]. Table 1 shows the Dsynt

I

MTKDRLAALHAAQSDDEEETEVAVNVDGHDSYMDDFFAQVF~IR~DKV

50

syn

IA

.-...TQE.RT.KDS.DDD-D.T.T..-R.RF..E..E

.......

F...I

47

syn

IB

.-...TQE.RS.KDS

.......

C.E.L

46

Dsynt

1 IA

/%E . . . . . .

syn

IB

SED..Q...Q

1

H..-R.HF..E..E

QD~SAILSAPQTDEKTKQEId~DLMADIKI(IIAICRVBBKLKGIE

syn

Dsynt

.......

R ......

AS.NP

.....

.....

A..NP

...........

E...E..S

I00 .... T..K..S...S..

T .....

QNIEQEEQQNKSSADLRIRKTEHSTLSRKFVEVMTEYNRTQTDYRERCKG

syn

IA

.S . . . . . G L . R

..........

Q ............

syn

IB

.S . . . . . G L . R

..........

Q ................

Dsynt

1

T..K..S...A..

S...A..S

...........

T.TSE...D...S..PAI.AS

syn

IB

...........

T.T..E..D...S.KLAI..DD.K.D..MT..A.NE..T

Dsynt

1

RHQD~MI(I~TSII~LIIDMFMDM~I~ILVE

.... DSSIS..A.

IA

• .SE.I...N..R

syn

IB

• .NE.I ...... R ...... V ..................

1

.........................

147 146

200 SE..T

S QGEMIDRIEYIIVEIIAMDMVQTA

syn

Dsynt

........

RIQRQLEITGRPTNDDELEKMLEEGNSSVFTQGIIMETQQAKQTLADIEA

IA

96

150

A..SK..D...D

syn

97

197 196

250

N .... V...EOR.

247

N...SV...ER.

246

TQDTKKALKYQSKARRKKIMT£r.ICr.TVr.GI~YVSSYF-M

291

syn

IA

VS ..... V .............

I..CV'I...II..TIGGI.G-

288

syn

IB

VS ..... V .............

I..CV...Wr...SIC.6TLGL

288

Fig. 3. Alignment of Drosophila syntaxin 1 and rat syntaxins 1A and lB. Alignment was obtained with the program CLUSTAL (PCGENE). Computation parameters were set as: K-tuple value 1; Gap penalty 5; Window size 10; Filtering level 2.5; Open gap cost 10; Unit gap cost 10. The alignment of the three sequences gave a global identity on 186 residues (63.7%) and similarity on 79 residues (27.1%). Positions where Drosophila syntaxin 1 and rat syntaxin 1A or 1B are identical are indicated with (.). Residues 194 to 267 in rat syntaxin 1A are thought to interact with VAMP proteins of synaptic vesicles [4]. Underlined and bold italic residues at the C-terminus are suppossed to form a transmembrane region. Underlined and bold residues (30-54, 75-100, 201-222 and 230-247) could form coiled-coil regions.

J.R. Cerezo et al. / Molecular Brain Research 29 (1995) 245-252

249

the Purkinge cell dendrites and parallel fibers are not yet formed, shows no staining at the molecular layer (Fig. 6A). The staining of P15 cerebellum (Fig. 6C), once these synapses are formed, is present throughout the molecular layer, not labeling the cell bodies at the Purkinge, internal and external granular layer. At an intermediate time (P9), when these synapses are restricted to the lower molecular layer [13], the label is restricted to this area (Fig. 6B).

The transmembrane segment of Dsyntl is classified as multimeric according to the algorithm described by Eisenberg et al. [8], a fact that could be significant for the possible role of these molecules in forming a fusion channel previous to the release of neurotransmitter [11]. There is also a deviation from the general homology between these molecules at their N-terminal ends. The rest of the molecule shows a strong similarity between all three sequences including the location of predicted coiled-coil structures (see legend for Fig. 3) and the relative positions of possible casein kinase II, protein kinase C or c A M P / c G M P - d e p e n d e n t phosphorylation sites (Fig. 4).

4. Discussion

Using an antibody that recognizes a synaptic antigen in Drosophila and vertebrate nervous system we have cloned a eDNA coding the Drosophila homologue of rat syntaxin 1, a protein that could be involved in the docking a n d / o r fusion of synaptic vesicles to the actives zones of presynaptic plasma membrane-[ll]. As its vertebrate homologue Drosophila syntaxin 1 is neurospecific and located at synaptic areas of the CNS and synaptic boutons at the neuromuscular junction in the larva. The antibody used for expression cloning of Drosophila syntaxin 1 recognizes a neurospecific antigen in rat and human nervous system, which in SDSPAGE gels shows a similar mobility to rat syntaxin and a tissue distribution consistent with its synaptic location. There are slight differences in the relative mobility of the antigen recognized by mAb 44D5 in the three species studied (Fig. 1) and it seems that in the Drosophila homogenate there could be more than one band of similar mobility reacting with the antibody. It is possible therefore that mAb 44D5 recognizes more than one isoform of syntaxin. Nevertheless, both in Drosophila and rat, the antigen is only expressed in neural tissue indicating that the recognition by mAb 44D5 would be limited to the neurospecific syntaxin 1A or 1B isoforms [2]. Rat syntaxins 1A and 1B seem

3.4. Synaptic location of Dsyntl protein As indicated above, mAb 44D5 recognizes an antigen expressed in the neuropil of larval CNS. We further explored throughout development the location of the protein recognized by the antibody to confirm its synaptic location. In the embryo, staining is first observed in the neuropil during stage 17, coinciding with the maturation of synapses in the larval CNS (Fig. 5A). The staining persists in the CNS during the different larval instars (not shown) and in the adult it accumulates also in the neuropil of the brain not labelling the cell bodies; thus, in the adult CNS, the staining is resticted to the area of synaptic contacts between central neurons (Fig. 5B). Staining of larval muscles shows unequivocally the precise location of the protein at the synaptic boutons (Fig. 5C). The neural specific antigen recognized by mAb 44D5 has a similar molecular weight in all three species studied (Fig. 1) and it is also distributed in synaptic areas of the rat CNS. Fig. 6 shows the staining of rat cerebella of different postnatal ages (P4, P9 and P15). The spatial and temporal distribution of the label is consistent with the recognition of a synaptic antigen: P4 cerebellum, a time at which the synapses between

p2

1>2

C

N Rat syntaxln 1A

N

t

p3

I

pl p3

i

t

p3

i

p2 p3

i

I,,

p2p3

IL

p3

I

p2 p3

[

p3

,I

p2

,,I

m

I

C

Dsynt 1 Fig. 4. Potential phosphorylation and myristilation sites in rat syntaxin 1A and Dsyntl: (pl) cAMP- or cGMP-dependent phosphorylation, (p2) protein kinase C 1>hos1>horylation, (p3) casein kinase lI phosphorylation and (m) myristil group.

250

J.R. Cerezo et al. / Molecular Brain Research 29 (1995) 245-252

to be p r e s e n t in a m o l a r ratio of 2:1 a n d they are best s e p a r a t e d in T r i s - U r e a gels r a t h e r t h a n in the SDSP A G E gels, in which they r u n as a single b a n d (see Fig. 2 in ref. [26]). T h e s e results suggest that the a n t i b o d y against Drosophila syntaxin 1, used to clone the D s y n t l c D N A , recognizes the neurospecific m e m b e r s of rat syntaxins, that is o n e or both isoforms of rat syntaxin 1. T h e p r e s e n c e of a d o u b l e b a n d in larval CNS hom o g e n a t e s r u n in L a e m m l i gels (Fig. 1, lane 3) would suggest the existence in Drosophila of an h o m o l o g u e to each of the isoforms (1A a n d 1B) described in rat. T h e d e d u c e d a m i n o acid s e q u e n c e of D s y n t l is highly h o m o l o g o u s to rat syntaxin 1A (81.6%) a n d 1B

.6,

Fig. 5. Expression of Dsyntl during Drosophila development. A: immunohistochemistry performed with mAb 44D5 during embryogenesis shows protein accumulation in the neuropil, the lader-like structure of the CNS formed by bundles of axons, where most synaptic connections are established. B: in a transverse section of the adult head the protein is detected in the different neuropil areas of the brain, most notably in the neuropils of the medulla, lobula and lobula plate, which form the optic lobes. C: staining of the ventral musculature of the third instar larvae. Note the accumulation of Dsyntl in the synaptic boutons of the neuromuscular junction. Bars represent 40 /xm, 20 /xm and 10 /zm in panels A, B and C, respectively.

,n,

B

Fig. 6. Staining of rat cerebella from different postnatal ages with mAb 44D5. Parasagital cryostat sections, 25 /xm, of rat cerebella fixed and treated as described under Materials and methods. A: section of a postnatal day (P) 4 rat cerebellum. Notice the absence of immunoreactivity at the molecular layer. B: section of a P9 rat cerebellum. Notice the immunoreactivity as a thin band in the deepest part of the molecular layer. C: section of a P15 rat cerebellum. Notice the widening of the immunoreactive area in the molecular layer. Bars represent 20 #m in all three panels. (79.9%) a n d keeps the same similarity r e l a t i o n s h i p with o t h e r m e m b e r s of the rat syntaxin family as that observed for both isoforms of rat syntaxin 1. Based on these data, we can confidently suggest that the seq u e n c e here described as D s y n t l c o r r e s p o n d s to the Drosophila h o m o l o g u e of rat syntaxin 1. Nevertheless,

ZR. Cerezo et al. /Molecular Brain Research 29 (1995) 245-252

we have no evidence to decide whether the c D N A cloned would correspond to syntaxin 1A or 1B, if both isoforms were to be present in Drosophila. A number of synaptic proteins have been identified and sequenced in Drosophila which show a high similarity with their rat homologues, suggesting a precise conservation of the basic mechanisms for neurotransmitter release. Drosophila and rat syntaxins 1 show an even higher degree of conservation than that previously reported for other synaptic proteins: synaptotagmin [20], SNAP 25 [22] and synaptobrevin or neuronal vecicle associated membrane proteins (n-VAMPs) [7]. This suggests the existence of a high degree of functional similarity for syntaxin 1 in both species and possibly in humans, where the antibody used to clone Dsyntl recognizes a protein of similar size. As other members of the syntaxin family, Drosophila syntaxin 1 is an integral membrane protein containing 291 residues and a C-terminal segment associated with the membrane. In contrast to rat syntaxins 1A and 1B and according to the algorithm designed by Eisenberg et al. [8], the membrane associated C-terminal segment of Drosophila syntaxin 1 is classified as multimeric, a fact that could have implications for its possible function as a fusion channel or for its presumed interactions with other proteins of the presynaptic membrane. Although rat syntaxins 1A and 1B are classified by the program used as monomeric, the hydrophobic moment (/xH) values for their integral membrane C-terminal segments are close to the limit values for a multimeric protein. It is possible therefore that, in this regard, the three proteins could be functionally similar. The similarity of Drosophila syntaxin 1 with its rat homologues (syntaxin 1A and 1B) is highest at the internal part of the molecule, and this includes the location of possible coiled-coil structures, likely to be involved in protein-protein interactions, and the relative positions of potential phosphorylation sites. As it happens in other proteins of the rat syntaxin family, the region immediately adjacent to the C-terminal transmembrane segment contains one of these putative helical coiled-coil structures. It has been recently suggested that this region could serve for the interaction of syntaxin with the synaptic vesicle associated membrane proteins (VAMPs) 1 and 2 [4]. The high degree of conservation, in this and other regions of possible interaction with o t h e r proteins, suggests that Drosophila syntaxin 1 would interact with the corresponding homologues of other synaptic proteins. The phosphorylation of synaptic proteins by casein kinase II [3] could be implicated in the presynaptic events that take place after long term potentiation [1,5]. In this respect, the conservation of the relative position of potential phosphorylation sites by casein kinase II, protein kinase C and c A M P / c G M P dependent phosphorylation, particularly in the C-terminal

251

half of the molecule (Fig. 4), suggests that rat syntaxin 1 and Dsyntl could be functionally analogous not only in their interactions with other molecules of the synaptic complex, but also regarding the possible regulation of these interactions via phosphorylation. The high homology between Drosophila, rat and possibly human syntaxin 1 indicates the conservation of the basic mechanisms of synaptic release in different species. The cloning of Dsyntl opens the possibility to perform mutation analysis in this gene in order to understand synaptic function regulation. A similar strategy has started to be productive in the case of synaptotagmin: embryos that lack the syt gene fail to hacht and show an increase in the frequency of spontaneous miniature excitatory junctional potential (EJPs) with a reduction in the number and amplitude of evoked EJPs in the presence of Ca 2+, suggesting a key role of synaptotagmin in neurotransmitter release. [15]. Autoantibodies to p65 or syntaxin could be involved in the development of the Lambert-Eaton myastenic syndrome, appearing in patients with small cells lung cancer (SCLC) known to express abundantly both proteins [6]. Future genetic studies about Dsyntl function in Drosophila could also have important implications in the understanding of this condition.

Acknowledgements We would like to thank Consuelo Ferrer for assistance with nucleotide sequencing and Francisco Tejedor for help with larval neuromuscular preparations. The technical assistance of Rosa Garcia Velasco with tissue culture is acknowledged. This work was supported with Grants number PM88-0182 from D G I C Y T to F.M. and PB 90-0082 from D G I C Y T to F.J.

References [1] Bennett, M.K., Calakos, N. and Scheller, R.H., Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science, 257 (1992) 255-259. [2] Bennett, M.K., Garcia-Arraras, J.E., EIferink, L.A., Peterson, K., Fleming, A.M., Hazuka, C.D. and Scheller, R.H., The syntaxin family of vesicular transport receptors, Cell, 74 (1993) 863-873. [3] Bennett, M.K., Miller, K.G. and Scheller, R.H., Casein kinase II phosphorylates the synaptic vesicle protein p65, J. Neurosci., 13 (1993) 1701-1707. [4] Calakos, N., Bennett, M.K., Peterson, K.E. and Scheller, R.H., Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking, Science, 263 (1994) 1146-1149. [5] Charriaut-Marlangue, C., Otani, S., Creuzet, C., Ben-Ari, Y. and Loeb, J., Rapid activation of hippocampal casein kinase I1 during long-term potentiation, Proc. Natl. Acad. Sci. USA, 88 (1991) 10232-10236. [6] David, P., El Far, O., Martin-Mouto, N., Poupon, M.F., Takahashi, M. and Seagar, M.J., Expression of synaptotagmin and

252

J.R. Cerezo et aL / Molecular Brain Research 29 (199.5) 24.5-252

syntaxin associated with N-type calcium channels in small cell lung cancer, FEBS Lett., 326 (1993) 135-139. [7] DiAntonio, A., Burgess, R.W., Chin, A.C., Deitcher, D.L., Scheller, R.H. and Schwarz, T.L., Identification and characterization of Drosophila genes for synaptic vesicle proteins, J. Neurosci., 13 (1993) 4924-4935. [8] Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R., Analysis of membrane and surface protein sequences with the hydrophobic moment plot, J. Mol. Biol., 179 (1984) 125-142. [9] Hata, Y., Slaughter, C.A. and Sudhof, T.C., Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin, Nature, 366 (1993) 347-351. [10] Huynh, T.V., Young, R.A. and Davis, R.W., Constructing and screening cDNA libraries in Agtl0 and Agtll. In D.M. Glover (Ed.), DNA Cloning-A Practical Approach, Vol. 1, IRL Press, Oxford, 1985, pp. 49-78. [11] Jahn, R. and Siidhof, T.C., Synaptic vesicles and exocytosis, Annu. Rev. Neurosci., 17 (1994) 219-246. [12] Laemmli, V.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. [13] Larramendi, L.M.H., Analysis of synaptognesis in the cerebellum of the mouse. In R. Llin~s (Ed.), Neurobiology of Cerebellar Evolution and Development. Proceedings of the First International Symposium of the Institute for Biomedical Research, American Medical Association, Chicago, 1969, pp. 803-843. [14] IAvEque, C., El Far, O., Martin-Moutot, N., Sato, K., Kato, R., Takahashi, M. and Seagar, M.J., Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis, J. Biol. Chem., 269 (1994) 6306-6312. [15] Littleton, J.T., Stern, M., Schulze, K., Perin, M. and Bellen, H.J., Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release, Cell, 74 (1993) 1125-1134. [16] Llimls, R., Sugimori, M. and Silver, R.B., Microdomains of high calcium concentration in a presynaptic terminal, Science, 256 (1992) 677-679. [17] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [18] Mart~n-Bermudo, M.D., Gonz~lez, F., Domfnguez, M., Rodriguez, I., Romani, S., Modollel, J. and Jim6nez, F., Molecu-

lar characterization of the lethal of scute genetic function, Development, 118 (1993) 1003-1012. [19] O'Connor, V.M., Shamotienko, O., Grishin, E. and Betz, H., On the structure of the 'synaptosecretosome'. Evidence for a neurexin/synaptotagmin/syntaxin/Ca 2+ channel complex, FEBS Lett., 326 (1993) 255-260. [20] Perin, M.S., Johnston, P.A., Ozcelik, T., Jahn, R., Francke, U. and Sudhof, T.C., Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans, 3. Biol. Chem., 266 (1991) 615-622. [21] Pevsner, J., Hsu, S.-C. and Scheller, R.H., n-Secl: a neuralspecific syntaxin-binding protein, Proc. Natl. Acad. Sci. USA, 91 (1994) 1445-1449. [22] Risinger, C., Blomqvist, A.G., Lundell, 1., Lambertsson, A., Niissel, D,, Pieribone, V.A., Brodin, L. and Larhammar, D., Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones, J. Biol. Chem., 268 (1993) 24408-24414. [23] Sanger, F., Nicklen, S. and Coulson, A.R., DNA sequencing with chain-terminating inhibitors, Proc. NatL Acad. Sci. USA, 74 (1977) 5463-5467. [24] Snyder, M., Elledge, S., Sweetser, D., Young, R.A. and Davis, R.W., lgtll: gene isolation with antibody probes and other applications. In R. Wu and L. Grossman (Eds.), Methods in Enzymology, Vol. 154, Recombinant DNA part E, Academic Press, San Diego, CA, 1989, pp. 309-329. [25] S611ner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H. and Rothman, J.E., A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion, Cell, 75 (1993) 409-418. [26] S611ner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J.E., SNAP receptors implicated in vesicle targeting and fusion, Nature, 362 (1993) 318-324. [27] Stanley, E.F., Single calcium channels and acetylcholine release at a presynaptic nerve terminal, Neuron, 11 (1993) 1007-1011. [28] Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of protein from acrylamide gels to nitrocellulose sheets, Proc. Natl. Acad. Sci. USA, 76 (1979) 4350-4354. [29] Yoshida, A., Oho, C., Omori, A., Ito, T. and Takahashi, M., HPC-1 is associated with synaptotagmin and omega-conotoxin receptor, J. Biol. Chem., 267 (1992) 24925-24928.