Brain Research, 509 (1990) 1-7

1

Elsevier BRES 15177

Research Reports

Torpedo synaptophysin: evolution of a synaptic vesicle protein David Cowan, Michal Linial and Richard H. Scheller Department of Biological Sciences, Stanford University, Stanford, CA 94305 (U.S.A.) (Accepted 11 July 1989)

Key words: Synapse; Synaptic vesicle; Synaptophysin; Torpedo californica; Electric organ; Molecular cloning

Synaptophysin is an integral membrane protein of synaptic vesicles found in neurons and endocrine cells. Synaptophysin monomers associate into hexamers forming a large conductance channel. We present an analysis of synaptophysin from the nervous sytem of the marine ray Torpedo californica. Analysis of cDNA clones reveals a 62% amino acid similarity between the Torpedo and rat sequences. The 4 hydrophobic membrane spanning domains and the glycosylation site are conserved. In contrast, the two intravesicular loops connecting the membrane spanning regions, show varying degrees of sequence conservation, suggesting that portions of these domains may play critical functional roles. The carboxyterminal tail has been proposed to bind calcium and is a major site for tyrosine phosphorylation. The precise sequence of this region has almost completely diverged while the proline-tyrosine rich nature is maintained. Blotting studies reveal the RNA and protein in nervous system tissues and demonstrate that the molecule copurifies with cholinergic synaptic vesicles. INTRODUCTION Synaptic vesicles are major organelles of the nerve terminal responsible for uptake, storing, docking and regulating release of neurotransmitter. While these processes are central to brain function and likely sites for modulatory actions during learning, little is known about the molecular mechanisms of synaptic vesicle function. Synaptic vesicle associated proteins have been studied using biochemical, immunological and molecular genetic techniques. More recently vesicle proteins including synapsin 12'5'6'26 synaptophysin (p38) 3'13'21 and VAMP-1 (pl8, synaptobrevin) 1,24 have been characterized using molecular genetic techniques. Mammalian synaptophysin is a 38 kDa glycoprotein 9' ~5,16,28 which is 88% homologous between the human, bovine and rat amino acid sequences 1°. Based on the predicted amino acid sequence in conjunction with biochemical and immunological experiments a model for the m e m b r a n e topology of synaptophysin has been proposed m'2~. These data predict that the protein traverses the vesicle lipid bilayer 4 times with both the amino and carboxyterminal ends facing the cytoplasm. Evolutionary comparisons within mammalian species suggest that the m e m b r a n e spanning domains and the carboxyterminal tail are conserved while the loop regions are relatively free to diverge. Cross-linking experiments demonstrate that the protein associates into a homohexamer and upon reconstitution into a lipid bilayer a

channel with large unit conductance is produced 22. The carboxyterminal tail of synaptophysin is a site for tyrosine phosphorylation 17 and is believed to bind calcium 19. Synaptophysin may be involved in regulating the release of transmitter by forming a pore between the vesicle membrane and the presynaptic terminal 22 or may regulate the storage contents of vesicles. The densely innervated electric organs of marine rays have been used extensively as a source to purify cholinergic synaptic vesicles. These vesicles have a lipid to protein ratio of 5:1 and contain approximately 50,000100,000 molecules of acetylcholine as well as about 15,000 molecules of ATP 25'27. Recently, Rahamimoff et al. have shown that these vesicles contain a channel with a large conductance similar to that observed in lipid bilayer reconstruction experiments with mammalian brain synaptophysin Is. This channel has other similarities to the mammalian synaptophysin channel including a long opening time of up to a few seconds. In this report, we have used molecular genetic techniques to characterize a protein homologous to synaptophysin from the marin ray Torpedo californica. This protein is likely to comprise the channel observed in cholinergic vesicles from marine rays. The gene is specifically expressed in nervous system tissue and is predicted to encode a 301 amino acid protein which is 62% homologous to the mammalian proteins. The tyrosine and proline rich nature of the carboxyterminal portion of the Torpedo synaptophysin is conserved while

Correspondence: R. H. Scheller, Department of Biological Sciences, Stanford University, Stanford, CA 94305, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

the precise sequence has almost completely diverged. C o m p a r i s o n of the rat and m a m m a l i a n sequences allows a further definition of evolutionary conserved regions of the synaptophysin molecule which may delineate functional domains. MATERIALS AND METHODS

eDNA library screening and DNA sequencing 180,000 plagues of a lambda gt-ll cDNA library from Torpedo electric lobe were screened using a human synaptophysin3 probe containing the complete coding region (nucleotide 272-919). Hybridization and washing were carried out at 60 °C in 1× SSC. The positive clones were plaque purified and the cDNA inserts were subcloned in Bluescript KS vectors (Stratagene, La Jolla, CA). Plasmids were prepared by CsCI banding and were treated by the Exo-III, SI deletion method to produce overlapping clones at about 200 nucleotide intervals. Single stranded DNA from superinfected cultures were then subjected to sequence analysis using either the T7 or T3 primers.

RNA preparation and Northern blot analysis Total RNAs from different Discopyge ommata tissues were prepared by lysis in 4 M guanidine thiocyanate, layering over a cushion of 5.7 M CsCI, 0.1 M EDTA (pH 7.0), and centrifugation for 16 h at 30,000 rpm at 20 °C in a SW41 rotor4. RNA pellets were phenol extracted, precipitated and dissolved in water. RNA was quantitated by measuring absorbanee at 260 nm, and the integrity of the RNA was checked by staining. RNA samples were denatured in 50% formamide, 6% formaldehyde, 20 mM MOPS (pH 7.0), 1 mM EDTA at 55 °C for 15 min and fractionated on a formaldehydeagarose gel. RNA was blotted onto Nytran filters (Schleieher and Schull), and cross-linked under UV light. 32p probes were generated using hexanucleotide priming method7. Hybridizations were carried out at 42 °C for 18 h in 50% formamide, 5× SSPE (3 m Nall, 0.2 M NaH2PO4-H20 , 0.02 M EDTA, pH 7.4) 5x Denhardt's solution, 0.1% SDS and 100/~g/ml sheared denatured salmon sperm DNA. Blots were washed in 0.1 × SSC (3 M Nail, 0.3 M Na citrate, pH 7.4) 0.5% SDS at 68 °C for 2 h before exposed to X-ray film.

Western blot analysis Protein samples from either Torpedo californica tissues or from synaptic vesicle preparations were dissolved in Laemmli gel loading buffer (0.125 M Tris-C1 (pH 6.8), 2% SDS, 5% fl-mercaptoethanol and 0.025% bromophenol blue), and fractionated on a 10% SDS-polyacrylamide gel12. The gels were transferred to nitrocellulose filters in Tris-glycine buffer, 20% methanol23. After transfer the filters were air dried, washed several times with PBS, and blocked for 1 h in PBS containing 5% BSA, 0.05% Tween-20 and 5% normal goat serum (PBTS). The filters were incubated for 12-18 h at 4 °C with the primary antibodies (in 1:1000 dilution), in PBTS buffer. All subsequent incubations were carried out at room temperature. The filters were washed 4 times (10 min each) in PBS, 0.1% BSA, 0.05% Tween-20 (PBT), and incubated for 2-4 h with 0.1 ~Ci/ml iodinated~25I goat anti-rabbit IgG (ICN Radiochemical, CA). RESULTS

Torpedo synaptophysin A radioactively-labeled h u m a n synaptophysin c D N A clone was used to screen l a m b d a gt-ll c D N A clones constructed using poly A ÷ m R N A from electric lobe of Torpedo californica at 60 °C. A single positive recombinant out of a p p r o x i m a t e l y 180,000 plaques was purified and analyzed further. The nucleotide sequence of both

strands of the coding region was d e t e r m i n e d by the dideoxy chain termination m e t h o d and is presented in Fig. 1. The clone contained a b o u t 1500 nucleotides of 5"-untranslated sequence at least part of which m a y be due to the ligation of two c D N A s during the p r e p a r a t i o n of the c D N A library. Since the protein coding region is homologous throughout, this does not effect our analysis and much of this non-coding sequence is not presented. The open reading frame predicts a protein of 30l amino acids with th~ same general hydrophobicity pattern as m a m m a l i a n synaptophysin. The h o m o l o g y to the m a m m a l i a n sequences is p r e s e n t e d in Fig. 2. While the 3 m a m m a l i a n species differ at a total of 26 position there are 114 amino acid substitutions b e t w e e n the Torpedo and rat sequences. Both the length and the spacing between the 4 t r a n s m e m b r a n e domains is maintained while in the c a r b o x y t e r m i n a l cytoplasmic region 3 small insertion/deletions are n e e d e d to maximize the extent of homology. In the m a m m a l i a n synaptophysins only a single substitution is located within the m e m b r a n e spanning domains while the Torpedo sequence diverges at 20 positions when c o m p a r e d to rat. O n l y 4 of these substitutions are not conservative and isoleucine, leucine, valine and methionine seem to substitute for each o t h e r rather freely. T h r e o n i n e to serine (postition 42) and serine to threonine (position 55) substitutions toward the e n d of the first t r a n s m e m b r a n e d o m a i n (M I) suggests that the polar nature of these residues m a y need to be m a i n t a i n e d for the p r o p e r function of the molecule. In general, the charged residues which flank the m e m b r a n e spanning domains are conserved; the type of substitution o b s e r v e d being arginine to lysine as in position 132. C o m p a r i s o n s of the 3 m a m m a l i a n synaptophysin sequences suggests the two loop regions facing the lumen of the vesicles are relatively free to diverge, suggesting little functional constraint on the sequence. A l t h o u g h comparison of rat and Torpedo reveals 37 substitutions in these loop regions stretches of sequence do a p p e a r to be highly m a i n t a i n e d between species. For instance, the sequences of the loop regions within 5 - 1 0 amino acids of the m e m b r a n e spanning domains are m o r e highly conserved than most internal regions of the loops (Fig. 3). The longest stretch of perfectly matching sequence (14 residues) is the a m i n o t e r m i n a l region of the forth m e m b r a n e d o m a i n (M IV). Interestingly, within the l o o p connecting M I and M lI a strongly conserved region (residues 65-76) contains the second longest stretch of perfect h o m o l o g y between rat and Torpedo sequences. The carboxyterminus of synaptophysin consists of about 80 amino acids which extend into the cytoplasm (Fig. 3). This portion of the protein contains a n u m b e r of tyrosines which serve as sites for a protein kinase 17. In the

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Fig. l. Nuclcotid¢ and predicted amino acid sequence of Toledo synaptophysin. The nucleic acid sequence is numbered on the left and the predicted amino acid sequence on the right. Amino acids arc given using the single letter code below the DNA sequence.

mammalian synaptophysins the carboxyterminal region consists of 10 non-identical copies of the core sequence tyrosine-glycine-proline/glutamine-glutamine-glycine 21. The presence of a large number of proline residues

predicts a structure that is not compatible with an a helix. In comparing the Torpedo and mammalian sequences it is only possible to align this region of the protein to give a weak homology. Small stretches of 7 amino acids at

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Fig. 2. Amino acid sequences of Torpedo, rat, human and bovine synaptophysins. The complete predicted amino acid sequence of Torpedo synaptophysin is presented and the differences in the other species are written below 3"1°A3'2°'21. The regions comprising the 4 predicted membrane spanning domains are labeled above the sequence M I - M IV. Sequence not available is represented by ( . . . . ) and deletions in the sequences necessary to maximize alignment are indicated by ( . . . . ).

positions 265-273 are homologous as are the very carboxyterminal 8 amino acids. It is interesting to note that while the Torpedo and mammalian carboxyterminal sequences are not homologous in precise sequence the general character of the region is maintained. The mammalian carboxyterminus of synaptophysin has 9 tyrosine and 11 proline residues while the Torpedomaintains 8 tyrosine and

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13 proline residues in this region. The untranslated regions between the ray and mammals have completely diverged, retaining a similar composition yet no discernible homology. This includes an AT rich sequence which is 100% conserved in the mammalian synaptophysin cDNA sequences l° and absent in the Torpedo analog.

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ooo Fig. 3. Schematic of the amino acid substitutions in mammalian and Torpedosynaptophysin. Topology of synaptophysin molecule is taken from previous publications. The schematic on the right indicates the amino acid substitutions between the 3 mammalian species: rat, bovine, and human. The schematic on the right indicates the substitutions between the Torpedo and rat sequences. Closed circles indicate conserved substitutions using the following grouping of amino acids - - Met, Ile, Leu, Val; Phe, Tyr, Trp; Asp, Glu, Asn, Gin: Lys, Arg; His; Ala, Giy, Pro, Ser, Thr; Cys. Open circles indicate non-conserved changes. The proline (P) and tyrosine (Y) residues are indicated in the carboxyterminal cytoplasmic domain. The sugar addition site is indicated (2). A + indicates an additional amino acid and a - a deleted amino acid necessary for maximum homology.

Western blot of brain and lobe tissues while this protein is greatly enriched in purified synaptic vesicle fractions. In SDS/PAGE gels, this band corresponds to a major Coomassie blue staining protein that is specific to vesicles. The synaptophysin immunoreactive band is at or very near the position of another vesicle protein, VAT-1 when fractionated on a one dimension SDS-PAGE gel 14. Aminoterminal sequence analysis of the of electroeluted material migrating at 40-41 kDa indicates that the VAT-1 protein accounts for about 30% of the protein mass migrating at this position.

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DISCUSSION O

Fig. 4. RNA and protein blotting. Top: 12/~g of total RNA from various tissues of Discopygeommatawas fractionated on formaldehyde-agarose gels transferred to a Nytran filter and hybridized with a 3Zp labeled fragment containing the synaptophysin coding region. The filter was washed at 68 °C in 0.1 × SSC and exposed. Lane A, brain; B, electric lobe; C, spinal cord; D, electric organ; E, muscle; F, liver. RNA size markers are indicated in kilobases, Kb. Bottom: 10 ~g of protein from different tissues were fractionated by SDS-PAGE, blotted and probed using rat synaptophysin polyclonal antibodies. Lane A, rat brain; B, NGF induced PC-12 cells. Torpedotissues: Lane C, electric lobe; D, electric organ; E, muscle; F, purified synaptic vesicles; G, liver. Protein size markers are in kilodaltons, kDa.

Expression of synaptophysin in Torpedo The cloned Torpedo cDNA was used to probe the expression of synaptophysin in various tissues (Fig. 4). A 1.9 kb band was found in brain, electric lobe and spinal cord and no hybridization was seen in non-neuronal tissues including electric organ, muscle and liver. In mammalian species synaptophysin is expressed in endocrine tissues. This is likely to be the case in Torpedo as well, however, we have not investigated these tissues. Localization of the synaptophysin protein in Torpedo was studied using a serum antibody generated against the rat protein 21 (Fig. 4). A kDa band is observed in a

The divergence observed between the Torpedo and mammalian synaptophysin genes allows a prediction of functionally important regions of the molecule based on evolutionary conservation. Torpedo diverged from the mammalian lineage approximately 400-500 million years ago and during this time 114 substitutions have accumulated between Torpedo and rat while only 26 substitutions separate the rat, bovine and human synaptophysins. By including the Torpedo sequence in the comparisons, a refined and in some cases different set of conclusions are made regarding the evolutionary conservation of specific regions of the synaptophysin molecule. The conservation of short stretches of amino acid sequence flanking the transmembrane domains suggests that these regions may be necessary not only to anchor the membrane spans within the lipid bilayer but also to form appropriate environment around the opening of the pore. Both evolutionary comparisons and mutagenesis studies of the acetylcholine receptor have demonstrated that charged residues at the opening of the pore effect the conductance of the channel s . Another strongly conserved region includes the asparagine-histidine-serine glycosylation sequence in the Torpedo gene which is functionally conserved as an asparagine-lysine-threonine in the mammalian species. Thus, while the purpose of the sugars on synaptophysin is not known the conservation suggests these residues are important in some aspect of assembly, targeting or function. A highly conserved region in the center of the first loop is located just carboxyterminal to the N-linked glycosylation site. By the currently accepted model for synaptophysin topology it would lie inside the vesicle and outside of the nerve terminal membrane after vesicle fusion. In vitro mutagenesis of this region may provide a clue as to the possible function of this domain. The highest frequency of substitutions between Torpedo and mammalian synaptophysin occurs in the cytoplasmic tail portion of the protein. The high frequency of tyrosine, proline and glutamine (39% in rat and 43% in

Torpedo) is m a i n t a i n e d between species while the precise p r i m a r y sequence is altered, suggesting that strong functional pressures exist to maintain the composition of the sequence. Thus, it is likely that a rigid structure which has the ability to serve as a substrate for a tyrosine kinase are key features of this region, yet a precise structure and t h e r e f o r e sequence is not critical for the function of this d o m a i n of the protein. A l t e r n a t i v e l y , the fact that 5 out of 6 changes in the tail sequence between all m a m m a l i a n species are conserved might argue that compensating changes within interacting systems m a y have co-evolved allowing sepcific amino acid substitutions. A s a result of this divergent c a r b o x y t e r m i n a l sequence a monoclonal a n t i b o d y raised against this region of the rat protein 2~ does not react with the Torpedo molecule. In spite of the different a m i n o terminal sequences, prediction of the s e c o n d a r y structure by the C h o w - F a s s m a n algorithm reveals a similar p r e d i c t e d structure d o m i n a t e d by a r e p e a t unit of 4 - 6 amino acids. T h e c a r b o x y t e r m i n u s of synaptophysin has been pro-

posed to bind Ca 2+ with an affinity only about 4-fold lower than calmodulin 19. This is somewhat surprising in that the structure of synaptophysin does not fit into the known structural motif for calcium binding domains i~. The lack of precise sequence conservation further suggests that this d o m a i n may not form a precisely folded binding pocket for calcium. F u r t h e r , it has not yet been possible to d e m o n s t r a t e an effect of calcium on the channel p r o p e r t i e s of synaptophysin. The molecular cloning of c D N A s encoding the gene for synaptophysin from several species allows an evolutionary comparison suggesting i m p o r t a n t functional domains of the protein. The Torpedo sequence presented here provides further tools for investigating the structure and function of the synaptophysin gene in the electrom o t o r system, an enriched source of synapses. Acknowledgements. We would like to thank Regis Kelly and Kathy Buckley for the human synaptophysin cDNA and the laboratory of Paul Greengard for the rat synaptophysin antibody. M.L. is supported by a Chaim Weizman Fellowship.

REFERENCES 1 Baumert, M., Maycox, ER., Navone, E, Camilli, D.E and Jahn, R., Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain, EMBO J., 8 (1989) 379-384. 2 Browning, M.D., Huang, C.-K. and Greengard, P., Similarities between protein IIIa and protein IIIb, J. Neurosci., 7 (1987) 847-853. 3 Buckley, K.M., Floor, E. and Kelly, R.B., Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein, J. Cell Biol., 105 (1987) 2447-2456. 4 Chirgwin, J.M., Pryzybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 52945299. 5 De Camilli, P. and Greengard, P., Synapsin I: a synaptic-vesicle associated neuronal phosphoprotein, Biochem. Pharmacol., 35 (1986) 4349-4357. 6 De Camilli, R, Cameron, R. and Greengard, E, Synapsin I (protein I), a nerve terminal-specific phosphoprotein I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections, J. Cell Biol., 96 (1983) 1331-1354. 7 Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction fragments to high specific activity, Anal. Biochem., 132 (1983) 6-13. 8 Imoto, K., Methfessel, C., Sakmann, B., Mishina, M., Mori, Y., Konno, T., Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y. and Numa, S., Location of an a-subunit region determining ion transport through the acetylcholine receptor channel, Nature (Lond.), 324 (1986) 670-674. 9 Jahn, R., Schiebler, W., Ouimet, C. and Greengard, P., A 38,000 dalton membrane protein (p38) present in synaptic vesicles, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4137-4141. l0 Johnston, P.A., Jahn, R. and Sudhof, T.C., Transmembrane topography and evolutionary conservation of synaptophysin, J. Biol. Chem., 264 (1989) 1268-1273. 11 Kretsinger, R.H., Calcium coordination and the calmodulin fold: divergent versus convergent evolution, Cold Spring Harbor Syrup. Quant. Biol., 52 (1987) 499-510. 12 Laemmli, U.K., Cleavage of structural proteins during the

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