THE JOURNAL OF BIOLOCKXL CHEMISTRY Vol. 265, No. 27, Issue of September 25, pp. 16527-16533, 1990 Cc) 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed m U.S. A.

Synapsin II MAPPING OF A DOMAIN IN THE NH,-TERMINAL REGION WHICH BINDS TO SMALL SYNAPTIC VESICLES*

(Received for publication, April 12, 1990)

Gerald Thiel$$n, Thomas C. Siidhof$ll , and Paul Greengardt From the SLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021 and 6Howard Hushes Medical Institute and II Denartment of Molecular Genetics, University of Texas Southwestern Medical School, &las, Texas 75235

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The synapsins are a family of neuron-specific phos- phoproteins that selectively bind to small synaptic ves- icles in the presynaptic nerve terminal. Using the cDNA encoding rat synapsin IIb, we employed an Esch- erichia coli expression system to synthesize a variety of fusion proteins containing a truncated protein A linked to different portions of the NH&erminal region of synapsin II. The recombinant proteins were purified by IgG-Sepharose chromatography and tested in vitro for their ability to bind to purified synaptic vesicles. These experiments identified a region between amino acids 43 and 121 of the amino-terminal portion of synapsin II which binds to synaptic vesicles. Mild tryp- sinization of synaptic vesicles reduces binding of re- combinant proteins to synaptic vesicles, suggesting that the interaction between synapsin II and the vesi- cles is in part mediated by a synaptic vesicle protein. The 42 NHz-terminal amino acids of synapsin II are not necessary for binding to synaptic vesicles, although this domain contains the phosphorylation site for CAMP-dependent protein kinase.

The synapsins are a family of four synaptic vesicle phos- phoproteins that contain homologous amino-terminal do- mains and different combinations of distinct carboxyl-termi- nal domains (Siidhof et al., 1989b). Synapsin I consists of two closely related proteins termed synapsin Ia and synapsin Ib, with apparent molecular weights of 86,000 and 80,000, respec- tively, on SDS-PAGE.’ Synapsin II consists of two closely related proteins, termed synapsin IIa and synapsin IIb, with apparent molecular weights of 74,000 and 55,000, respectively, on SDS-PAGE. All four synapsins are substrates for protein kinases: CAMP-dependent protein kinase and Ca’+/calmodu- lin-dependent protein kinase I phosphorylate both synapsin I and II while only synapsin I is a substrate for Ca’+/calmodu- lin-dependent protein kinase II (Huttner and Greengard, 1979; Huttner et al., 1981; Nairn and Greengard, 1987).

A variety of physiological stimuli affecting synaptic func-

* This work was supported by United States Public Health Service Grants MH 39327 and NS 21550 and Contract 813826 with the United States Environmental Protection Agency. The costs of pub- lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- me&” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Support,ed in part by the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed.

’ The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HEPES, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; bp, base pair; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid.

tion have been shown to regulate the state of phosphorylation of synapsins I and II (for review, see Nestler and Greengard, 1984; De Camilli and Greengard, 1986). Phosphorylation of synapsin I by Ca’+/calmodulin-dependent protein kinase II decreases the binding of synapsin I to synaptic vesicles (Schie- bler et al., 1986; Benfenati et al., 1989b). This correlation between the phosphorylation state of synapsin I and its bind- ing to synaptic vesicles led to the hypothesis that synapsin I might be involved in the regulation of neurotransmitter re- lease. Injection studies using the squid giant synapse provided direct evidence in support of this concept (Llinas et al., 1985).

Less is known about the properties and functional role of synapsin II. Recent cloning of cDNAs of all four synapsins demonstrated that synapsins I and II share a common amino- terminal region with an overall sequence identity of 70% covering 420 residues (Siidhof et al., 1989b). The sequences of synapsins I and II diverge in the COOH-terminal region. Like synapsin, I, synapsin II is localized on small synaptic vesicles in the presynaptic nerve terminal, but a detailed investigation of its vesicle-binding properties has not been carried out, mainly because the protein has been purified only in small amounts.

In a recent study of the binding of synapsin I to small synaptic vesicles, two different sites of interaction between synapsin I and synaptic vesicles were found. The NH,-ter- minal head domain penetrates the lipid bilayer and interacts with lipids. The COOH-terminal tail domain appears to bind to a protein component of the vesicle membrane (Benfenati et al., 1989a, 1989b). Because of the synapsin-lipid interaction, it was not possible in that study to examine whether the head domain of synapsin I also binds to a protein component on synaptic vesicles. However, such an interaction was proposed as a possible explanation for the specific subcellular localiza- tion of both synapsin I and synapsin II. Synapsin IIb is virtually equivalent to the head region of synapsin I and therefore can be considered a naturally occurring variant of synapsin I.

As one approach to investigate the binding of synapsin II to synaptic vesicles, we have expressed different fragments of synapsin II in bacteria as a fusion protein with a truncated protein A. Our results indicate that the NH, terminus of synapsin II contains at least one binding site for synaptic vesicles, which is sensitive to trypsin, suggesting that synapsin II interacts with a protein component on the vesicles. A preliminary account of this study has been presented (Thiel et al., 1989).

EXPERIMENTAL PROCEDURES

Materials-[y-“2P]ATP (2900 Ci/mmol), ‘2”I-labeled protein A (90 &i/pg), and NalzSI were from DuPont-New England Nuclear; IODO- BEADS were from Pierce Chemical Co.; ultra pure sucrose was from

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16528 Binding of Synapsin II to Synaptic Vesicles

Bethesda Research Laboratories, and nitrocellulose sheets (pore size 0.2 pm) were from Schleicher & Schuell. Restriction enzymes, DNA ligase, and Klenow fragment were purchased from New England Biolabs, Asp-718 was from Boehringer (Indianapolis, IN), goat serum and 4-chloro-1-naphthol were from Sigma. Other reagents were from the following sources: anti-protein A antibodies (Sigma): antipeptide antibodies specific for the NHz-terminal phosphorylation -site of synapsins I and II (gift of A. J. Czernik, The Rockefeller University); polyclonal antibodies against synaptophysin (Jahn et al., 1985); monoclonal antibody against p65 (Matthew et al., 1981; kindly pro- vided bv Dr. L. Reichardt, Universitv of California. San Francisco): peroxidase conjugated goat anti-rabbit IgG (Cappel, Malvern, PA) and alkaline phosphatase coupled goat anti-mouse antibodies (Pro- mega, Madison, WI). Catalytic subunit of CAMP-dependent protein kinase was gift of A. Horiuchi and A. Nairna (The Rockefeller University). Synapsin I was purified under nondenaturing conditions from rat brain (Bahler and Greengard, 1987). Synapsin II was purified by a monoclonal antibody affinity column (gift of Y-L. Siow, The Rockefeller University).

Plasmid Construction-To produce protein A-synapsin II fusion proteins we used the prokaryotic expression vector pRIT2T (Phar- macia. LKB Biotechnolopv Inc. Unpsala. Sweden). The plasmid pSKB; containing the fullrl”ength cDNA ofrat synapsin IIb ?Siidhof et al., 1989b), was digested with XhoI and NsiI, and the XhoI site was filled in with dCTP, dTTP, and the large fragment of DNA-polym- erase I. The 594-base pair fragment was isolated and cloned in the PstI site and BamHI site of the pRIT2T-vector, which was filled in with dGTP and dATP, leading to the plasmid pRIT2TSyl. To construct pRIT2T-Sy2 we isolated a 384-base pair XhoI-Sal1 frag- ment of pSKB, partially filled the XhoI site with dCTP and dTTP, and cloned it in the same sites of the wild-type vector as described for pRIT2TSyl. The newly generated plasmid pRIT2TSy2 was digested with NaeI and Sal1 and filled in with the Klenow fragment in the presence of dNTPs. The NueI-Sal1 fragment was removed on a 5% polyacrylamide gel and the cut vector isolated and religated. This plasmid, pRIT2TSy3, contains the 233-pb XhoI-NaeI fragment of pSKB, fused in frame with the truncated protein A sequence. The same strategy was used to create pRIT2TSy4: the plasmid pRITZT- Sy2 was digested with Asp-718 and Sal1 and filled in with the Klenow fragment in the presence of dNTPs. The Asp-718.Sal1 fragment was removed by polyacrylamide gel electrophoresis and the cut vector isolated. Religation led to plasmid pRITZTSy4, containing the 149. bp XhoI-Asp-718 fragment of pSKB. Plasmid pRIT2T-Sy5 was made bv inserting the 150-bp NaeI-Sal1 fragment of pSKB into the filled- in BamHI site and into the Sal1 site of the wild-type pRIT2T vector. Finally we isolated the 210-bp PuuII-Sal1 fragment of pSKB and cloned it into the SmaI and Sal1 site of pRITPT, creating plasmid pRIT2T-Sy6. Two pRIT2T constructs containing residues 647-704 of synapsin Ia and 647-668 of synapsin Ib, respectively, were made by polymerase chain reaction (Saiki et al., 1985) using oligonucleo- tides corresponding to nucleotides 2052-2080 on the sense strand and nucleotides 1937-1963 on the antisense strand, which contained an additional EcoRI site (sense-oligo) and BclI site (antisense-oligo). For the amplification reaction we used synapsin Ia and synapsin Ib cDNA as templates and a Perkin-Elmer thermocycler with denaturing tem- perature of 95 “C for 1 min and extension temperature of 58 “C for 10 min. The amplification product was purified, digested with EcoRI and BclI, and cloned in the EcoRI and BamHI site of pRIT2T. The plasmid pRIT2T-38e, which contains nucleotides 740-1021 from the rat synaptophysin cDNA, was described previously (Johnston et al., 1989).

All fusion proteins were constitutively expressed in Escherichia coli DH5n cells.

Purification of Recombinant Protein A-Synapsin II Fusion Pro- teins--For large scale protein purification, cells were grown overnight in LB medium (1% Bact,o tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) containing 100 Kg/ml ampicillin. Cells were harvested by centrifugation (30 min at 1,560 X g,.,, GS-3 rotor) and resuspended in l/10 volume of phosphate-buffered saline/Tween 20 (PBST: 50 mM phosphate buffer (pH 7.4), 150 mM NaCl, 0.05% Tween 20) containing 0.1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 10 pg/ml antipain, 2 fig/ml pepstatin, and 2 rg/ml chymostatin. The cells were disrupted by sonication on ice for 4 x 20 s at setting 8 (Microson Ultrasonic Cell Disruptor, Heat Systems, Ultrasonics, Inc., Farmington, NY), and the lysate was centrifuged for 10 min at 10,000 x g,,, at 4 “C in an SS 34 rotor, the supernatant was collected, diluted with 1 volume of PBST, and loaded onto an IgG-Sepharose column (Pharmacia). After washing with PBST and phosphate-buffered sa-

line the bound protein was eluted with 0.5 M acetic acid (pH 3.0), and dialyzed against 10 mM Tris/HCl (pH 7.6), 0.1 mM EDTA, and 1 mM 2-mercaptoethanol at 4 “C. The proteins were concentrated with Aquacide II (Calbiochem) and stored at -80 “C.

In Vitro Phosphoryhtion-The recombinant protein A-synapsin II fusion proteins were phosphorvlated in uitro bv the catalvtic subunit of CAMP-dependent protein kinase in a 50-~1 assay mixture contain- ing 50 mM HEPES (DH 7.3). 10 mM MeCL. 0.5 mM EGTA. 1 mM diihiothreitol, 3 pM ATP (specific activity-5 mCi/pmol), and 3 pg substrate protein, except in the case of rat synapsin II, where 1 rg was used. The reaction was started bv adding 1 rrl of protein kinase (1 mg/ml), carried out for 1 min at 30 “C, and stopped with 25 ~1 of SDS&on solution (125 mM Tris/HCl (DH 6.8). 3 mM EDTA. 20% (w/v) glicerol, 9% SDS, 10% 2-mercapioethanol, and 0.05% brom- phenol blue). To phosphorylate the recombinant proteins to full stoichiometry, we increased the ATP concentration to 150 PM and incubated for 30 min at 30 “C. A trace amount of [-y-“‘P]ATP was added to the reaction mixture to calculate the stoichiometry. The phosphorylated proteins were exhaustively dialyzed to remove free ATP.

Purification of Small Synaptic Vesicles and Liver Microsomes- Small synaptic vesicles were purified from rat neocortex (male Sprague-Dawley; body weight 150-200 g) through the step of chro- matography on controlled pore glass as described by Huttner et al. (1983). Endogenous synapsin I and about half of endogenous synapsin II were removed by incubation with 0.2 M NaCl (final concentration) as described (Benfenati et aZ., 1989a). The stripped vesicles were resuspended in glycine buffer (0.3 M glycine, 5 mMHEPES (pH 7.4), and 0.02% NaN3) and kept at 4 “C for 24 to 48 h prior to use. This additional incubation resulted in removal of virtually all remaining synapsin II from the vesicles, as tested by Western-blotting. Liver microsomes were nrenared as described (Thiel and Soling. 1988). The final 100,000 X g ,,,.: pellet was stripped with 0.2 MNaCl (final concentration) and the stripped microsomes resuspended in glycine buffer.

Limited Proteolysis of Small Synaptic Vesicles-Stripped synaptic vesicles were pelleted by centrifugation for 1 h at 350,000 X g,,,., using the TLA-100.2 rotor (Beckman Instruments). The pellet (approxi- mately 1 mg of protein) was resuspended in 400 ~1 of 50 mM HEPES (pH 7.4) and 200 mM glycine and incubated for 1 h at room temper- ature with N-tosyl-L-phenylalanine chloromethyl ketone-trypsin (trypsin/vesicle protein-weight ratio = l:l,OOO). An excess of soybean trwsin inhibitor (10 &ml) and 0.2 mM phenvlmethylsulfonyl fluo- ride were added to stopthe proteolysis, and the samples were further incubated for 30 min at 4 “C. The trypsinized vesicles were then centrifuged at 350,000 X g,,, for 1 h and washed three times in 5 mM HEPES (pH 7.4), 500 mM glycine, 10 rg/ml soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, incubated overnight at 4 “C in this buffer, and finally resuspended in glycine buffer for binding experiments.

Binding of Recombinant Protein A-Synapsin II Fusion Proteins to Synaptic Vesicles and Liver Microsomes-The proteins to be tested for binding were dialyzed overnight at 4 “C against 25 mM Tris/HCl (DH 7.4) and 175 mM NaCl. and centrifuged for 30 min at 4 “C at 350,000’~ g,,,,,. Due to the hydrophobicity of most of the recombinant proteins, a significant amount sedimented under these conditions. The supernatant was used for the binding assay. The assay was carried out in a volume of 100 ~1 containing 250 mM glycine, 30 mM NaCl, 5 mM Tris/HCl, 4 mM HEPES (pH 7.4), and 10 pg of synaptic vesicle or microsomal protein. The prespun recombinant proteins were added at. a concentration of 70 to 100 nM (depending on the proteolvsis). The samples were incubated on ice for 60 min and the bound protein was separated from the free protein by centrifugation for 30 min at 350,000 X g,.,. The clear yet visible vesicle pellet was resuspended in 50 ~1 of glycine buffer to which 25 ~1 of SDS-stop solution was added. The nroteins were resolved by 11% PAGE, transferred to a nitrocelluldse filter, and probed with-either an anti- peptide-antibody against synapsin phosphorylation site I or a poly- clonal anti-protein A antibody, followed by iz51-labeled Staphylococcus aureus protein A (2,000 cpm/lO ~1). The filters were air dried and exposed to x-ray film for 6-10 h. The two antibodies gave similar results. The recovery of the vesicles after centrifugation was tested by using iodinated vesicles. No radioactive proteins were found in the supernatant after spinning for 30 min at 350,000 X g,...

Miscellaneous Techniques-SDS-PAGE was performed according to Laemmli (1970). Gels were silver-stained following the procedure of Morrisey (1981). Protein was determined according to Lowry et al. (1951) or Bradford (1976). Synaptic vesicles were iodinated as follows.

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Binding of Synapsin II to Synaptic Vesicles 16529

The vesicles, kept in glycine buffer, were incubated with five pre- washed IODO-BEADS and 2.5 mCi of [“‘I]NaI. After incubation for 15 min at room temperature, the free iodine was removed by gel filtration on a 0.5 x lo-cm Sephadex G-50 column. The radioactive vesicle fractions were concentrated by centrifugation and finally resuspended in glycine buffer.

RESULTS

Expression of Synapsin II Fragments as Fusion Proteins with Protein A-The plasmid vector pRIT2T encodes a trun- cated form of protein A (residues 23-269) which contains four consecutive IgG-binding domains (Nilsson et al., 1985). The vector can express inserted DNA sequences fused to the COOH terminus of protein A under control of the phage PR promoter. The fusion proteins expressed from pRIT2T do not contain the protein A signal sequence and are therefore not secreted from the cells.

We decided to use this system to produce protein A-syn- apsin II fusion proteins because at present purified synapsin II is available only in limited amounts. Moreover, the pRIT2Tsystem promised high expression of recombinant proteins and provided an easy method for purification using the protein A portion of the protein and IgG-Sepharose as an affinity matrix. Purified proteins were needed because the aim of this study was to test the recombinant protein A- synapsin II fusion proteins for their ability to bind to synaptic vesicles in vitro and to serve as substrates for in vitro phos- phorylation reactions.

Fig. 1A shows the domain model of the synapsin family as proposed from the deduced primary structures and illustrated for synapsins Ib and IIb (Sudhof et al., 1989b). The large homologous region common to all synapsins (residues l-420) can be subdivided into three domains. Domain A (residues l- 29) contains the NHP-terminal phosphorylation site (site 1) followed by domain B (residues 30-112), which is less con- served between synapsin I and II. Domain C (residues 113- 420), the central homologous region, shows a high degree of homology between synapsins I and II.

Fig. lZ3 shows a diagram of part of the synapsin II cDNA with the restriction sites employed to obtain the different

A

B C

FIG. 1. Construction of protein A-synapsin II fusion pro- teins. A, diagram of the structure of synapsins Ib and IIb depicting the NH?-terminal domains A-C. The phosphorylation sites are marked. R, a restriction map of the 5’ portion of synapsin II cDNA. Coding sequences are represented by the open box. The restriction sites depicted here were used to construct the expression plasmids pRIT2TSy1, pRIT2TSy2, pRIT2TSy3, pRIT2T-Sy4, pRITZT- Sy5, and pRIT2T-Sy6, shown in C, which contained different frag- ments of the synapsin II cDNA fused in frame to a truncated protein A sequence.

expression constructs (pRIT2TSy1, pRIT2TSy2, pRITST- Sy3, pRIT2T-Sy4, pRIT2T-Sy5, and pRIT2TSy6). The NHp-terminal regions of synapsin IIa and synapsin IIb are identical and we therefore will not distinguish between these forms. For the construction of pRIT2T-Syl through pRITZT- Sy4, an XhoI site that is 25 nucleotides upstream of the initiator ATG was used. Therefore these constructs contain the 5’-coding region depicted in Fig. 1C and an additional 25 nucleotides of 5’-noncoding region which in the constructs serve as a linker between protein A and the amino terminus of synapsin II.

The pRIT2T-Syl product contains residues l-190 from synapsin II; it therefore includes the amino acid sequences of domains A and B and an additional 78 residues of domain C. The pRIT2T-Sy2 product consists of amino acids l-121 from synapsin II that represent domains A and B and 9 residues of domain C. The pRIT2T-Sy3 product contains amino acids l- 74 of synapsin II, which includes the domain A sequence and 45 residues of domain B. The protein encoded by pRITZT- Sy4 includes domain A and 13 residues of domain B. The gene products of pRIT2TSy5 and pRIT2TSy6 contain res- idues 75-121 and 51-121, respectively, of synapsin II. These protein A-synapsin II fusion proteins were constitutively ex- pressed in E. coli harboring the different plasmids.

The products of plasmids pRIT2T-Syl, pRIT2TSy2, pRIT2T-Sy5, and pRIT2T-Sy6 are clearly recognizable as major bands in gels containing total proteins (Fig. 2A, lanes 2,3, 6, and 7). The protein encoded by pRIT2T-Sy4 is visible as a faint band (Fig. 2A, lane 5; arrowhead), while the pRIT2T-Sy3 product can only be visualized on immunoblots (Fig. 2B). The expression of the truncated protein A encoded by the wild-type pRIT2T plasmid is shown in lane 1 of Fig. 2A (arrowhead). The apparent molecular weights of all recom- binant proteins were as predicted from their sequences. The recombinant proteins were mostly found in the particulate fraction in E. coli. However, using IgG-affinity purification the small amounts of soluble recombinant proteins could easily be purified. During purification, however, some protein A-synapsin II fusion proteins were proteolyzed in various degrees, despite the addition of protease inhibitors. Con- structs that contained larger fragments from the central ho- mologous domain of synapsin II produced completely insolu- ble proteins and were therefore not usable for our purpose (data not shown).

Phosphorylation of Protein A-Synapsin II Fusion Pro- teins-Synapsins Ia and Ib contain a phosphorylation site in the NH2 terminus (Ser-9) which serves as an acceptor for A B

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FIG. 2. Expression of protein A-synapsin II fusion proteins in E. coli. Total homogenate of E. co/i harboring the wild-type plasmid pRIT2T (lane I) and the constructs pRIT2TSyl (lone 2), pRIT2TSy2 (lane 3), pRIT2TSy3 (lane 4), pRIT2T-Sy4 (lane 5), DRITZT-Sv5 (lane 6). and DRITZT-Sv6 (lane 7) were senarated bv ‘SDS-PAG& and the gel stained with Coomassie.Blue (Af or blotted to nitrocellulose filters, probed with anti-protein A antibodies and developed using peroxidase-coupled goat anti-rabbit IgG antibodies (B). The immunblot contains 2 pg of protein/lane, ie. 10% of the protein shown in A.

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16530 Binding of Synapsin II to Synaptic Vesicles

CAMP-dependent protein kinase and for Ca*+/calmodulin- dependent protein kinase I (Czernik et al., 1987). Synapsins IIa and IIb contain a homologous sequence surrounding Ser- 10 and are also substrates for these kinases. We tested the different protein A-synapsin II fusion proteins for their ability to serve as substrates for CAMP-dependent protein kinase. All affinity-purified recombinant proteins containing the 42 NHz-terminal amino acids of synapsin II were substrates for CAMP-dependent protein kinase (Fig. 3). These are the pro- teins encoded by pRIT2T-Syl (lane 3), pRIT2TSy2 (lane 4), pRIT2TSy3 (lane 5), and pRIT2T-Sy4 (lane 6). The NH,- terminal 42 amino acids of synapsin II include three serine residues of which only one, Ser-10, contains the recognition sequence Arg-Arg-X-Ser(P) for CAMP-dependent protein ki- nase. In contrast, the products of plasmids pRIT2T-Sy5 and pRIT2T-Sy6 (Fig. 3, lanes 7 and 8), which lack Ser-10, could not be phosphorylated. For comparison, the phosphorylation of purified synapsins I and II is shown (Fig. 3, lanes 9 and 10). Phosphate incorporation into synapsins I and II as well as into the recombinant protein A-synapsin II fusion proteins containing residues l-42 was of the same order of magnitude. As negative controls we used the truncated protein A encoded by the wild-type pRIT2T plasmid and a protein A-synapto- physin fusion protein (Fig. 3, lanes 1 and 2). Neither of these proteins could be phosphorylated.

Phosphorylation of the pRIT2TSyl and pRIT2T-Sy3 gene products at higher ATP concentrations for 30 min led to an incorporation of 0.9 and 1.1 mol of phosphate/m01 of protein, respectively (data not shown). These results indicate that these fusion proteins can be stoichiometrically phosphoryl- ated in vitro.

Protein A-Synupsin ZZ Fusion Proteins Bind to Synaptic Vesicles-Synapsin II is present on small synaptic vesicles. However, the nature of this association and the binding site(s) are still unknown. We therefore studied the recombinant protein A-synapsin II fusion proteins for their ability to bind to purified stripped synaptic vesicles. We established a spe- cific binding assay using pRIT2T-Syl-encoded protein which contains the NH*-terminal 190 amino acids of synapsin II. Because the recombinant protein A-synapsin II fusion pro- teins were sometimes partially proteolyzed, we used Western blot technique rather than dot blots (Schiebler et al., 1986; Benfenati et al., 1989b) to measure the binding of intact

A B 1 2 3 4 5 6 7 S 9 10 1 2 3 4 5 6 7 8 9 10

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FIG. 3. Phosphorylation of affinity-purified protein A-syn- apsin II fusion proteins by CAMP-dependent protein kinase. E. coli harboring different pRIT2T expression plasmids were lysed by sonication and the expressed soluble fusion proteins were affinity- purified using IgG-Sepharose column chromatography. The purified proteins were phosphorylated in vitro by adding catalytic subunit of CAMP-dependent protein kinase as described under “Experimental Procedures.” For comparison, purified synapsins I and II were also analyzed. A, Coomassie Blue-stained gel; B, autoradiogram, illustrat- ing: truncated protein A (lane I); protein A-synaptophysin fusion protein (lane 2); the protein A-synapsin II fusion proteins encoded by pRIT2TSyl (lane 3), pRIT2TSy2 (lane 4), pRIT2T-Sy3 (lane 5), pRIT2TSy4 (lone 6), pRIT2T-Sy5 (lane 7), and pRIT2T-Sy6 (lane 8); synapsin I (lane 9) and synapsin II (lane IO). The band at 40 kDA in A is the exogenously added catalytic subunit of CAMP-dependent protein kinase (arrow).

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! FIG. 4. Binding of the pRIT2T-Syl gene product to intact

synaptic vesicles, trypsinized synaptic vesicles, and liver mi- &o&mes. Purified stripped synaptic vesicles, trypsinized synaptic vesicles. and liver microsomes were incubated with 100 nM uRITPT- Syl encoded fusion proteins. The bound protein was recovered by centrifugation, separated on SDS-PAGE, and blotted onto nitrocel- lulose filters. The blots were probed with an anti-protein A antibody, followed by lZSI-labeled S. aureuS protein A. The autoradiogram is depicted in the figure. Lane 1, no vesicles added (control); lone 2, intact synaptic vesicles; lanes 3 and 4, trypsinized synaptic vesicles using trypsin/vesicle protein weight ratios of 1:5000 and l:lOOO, respectively; lone 5, liver microsomes.

protein A-synapsin II fusion protein to synaptic vesicles. Fig. 4 shows that the product of pRIT2T-Syl sedimented with synaptic vesicles (lane 2). As a control, we carried out a parallel incubation without vesicles. The recombinant protein remained in the supernatant under these conditions (Fig. 4, lane 1).

To determine whether the binding of the pRIT2TSyl gene product was vesicle-specific and not the result of an interac- tion between the lipophilic membrane and a hydrophobic segment of the protein we undertook two approaches. First, we prepared liver microsomes in order to test whether a hydrophobic membrane of any origin is sufficient for binding the fusion protein. Fig. 4, lane 5, demonstrates that the protein encoded by the plasmid pRIT2T-Syl did not bind to liver microsomal membranes. We obtained comparable results using red blood cell ghost membranes (data not shown). Second, we incubated purified synaptic vesicles with low concentrations of trypsin and tested the ability of the recom- binant protein encoded by pRIT2T-Syl to bind to trypsinized vesicles. This experiment (Fig. 4, lanes 3 and 4) demonstrated that trypsinization of vesicles strongly reduced the binding of the protein encoded by plasmid pRIT2T-Syl. Therefore, the binding of this protein requires intact synaptic vesicles. We used iodinated vesicles to confirm that a 30 min spin at 350,000 x g,,,., was sufficient to pellet all labeled vesicle proteins from normal as well as trypsinized synaptic vesicles. Other samples containing intact or trypsinized vesicles were incubated with the recombinant protein but not centrifuged in order to assess the survival of the ligands in the incubation mixture. No residual protease activity could be observed (data not shown). Therefore, the reduced binding of the pRITZT- Syl encoded protein was due neither to reduced sedimentation of trypsinized vesicles nor to proteolysis of the ligand.

As a negative control, we tested the ability of the protein A-synaptophysin fusion protein to bind to synaptic vesicles. Synaptophysin is an integral membrane protein of small synaptic vesicles containing four transmembrane regions (Sudhof et al., 1987; Leube et al., 1987; Buckley et al., 1987). The protein A-synaptophysin fusion protein contains the carboxyl-terminal 73 amino acids of synaptophysin. As ex- pected there was no binding of this domain to synaptic vesicles (Fig. 5A, panel 38e). A nonspecific precipitation was observed, both in the absence and presence of synaptic vesicles. This result also shows that protein A does not bind to synaptic vesicles.

We tested the other recombinant protein A-synapsin II

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Binding of Synapsin II to Synaptic Vesicles 16531

FIG. 5. Binding of protein A-synapsin II fusion proteins to intact and trypsinized synaptic vesicles. A, the vesicle binding assay was performed as described under “Experimental Procedures” for the protein A- synaptophysin fusion protein (pRIT2T-38e) and the protein A-synapsin II fusion proteins encoded by the plasmids pRIT2TSy1, pRIT2TSy2, pRIT2T-Sy3, pRIT2TSy4, pRIT2T-Sy5, and pRIT2T-Sy6. For each recombinant protein three conditions are depicted: the left lane shows the control condition where no vesicles were added to the incubation mixture; the middle and right lanes show the binding to intact synaptic vesicles and trypsinized synaptic vesicles, respectively. A trypsin/vesicle protein weight ratio of 1:lOOO was used. B, the protein encoded by the plasmid pRIT2T-Sy3 was phosphorylated in vitro by CAMP-dependent protein kinase to a calculated stoichiometry of 1.1 mol of phosphate/m01 of protein and used in an in uitro vesicle binding assay. A comparison of the binding between the dephosphoform (lane I) and the phosphoform (lane 2) shows no difference at the concentration of 100 nM recombinant protein.

fusion proteins in order to narrow down the region of synapsin II necessary for binding to synaptic vesicles. The results of these binding experiments (depicted in Fig. 5A) demonstrate that in addition to the pRITZT-Syl gene product the proteins encoded by pRIT2TSy2, pRIT2T-Sy3, pRIT2TSy5, and pRIT2T-Sy6 could bind to synaptic vesicles. All of these proteins contain sequences of domain B of synapsin II. The results obtained with the fusion protein encoded by pRIT2T- Sy3 indicate that synapsin II contains a specific synaptic vesicle binding site in domain B in addition to the proposed binding site to lipids in domain C (Benfenati et al., 1989a, 1989b). The fact that the protein A-synapsin II fusion proteins encoded by pRIT2T-Sy3 and pRIT2TSy5 both bind to syn- aptic vesicles yet do not contain overlapping synapsin II sequences, suggests two binding sites in the synapsin II B region, but these may belong to one binding domain. The protein A-synapsin II fusion protein encoded by pRIT2T-Sy5 shows unspecific precipitation (panel Sy5, left lane), due to aggregation of the protein. However, the high degree of sedi- mentation due to binding to synaptic vesicles is clearly shown.

No specific binding was observed in experiments with the pRIT2T-Sy4 gene product. This protein contains the NH,- terminal 42 amino acids of synapsin II and includes the phosphorylation site (as shown in the previous section). From this result we can conclude that domain A of synapsin II does not bind directly to the vesicle membrane. We also tested two pRIT2T-encoded proteins, which contained the carboxyl-ter- minal portion of synapsin Ia (residues 647-668) and synapsin Ib (residues 647-668). Neither of these recombinant proteins bound to synaptic vesicles (data not shown).

Taken together these results indicate that fusion of short stretches of domain B of synapsin II to the protein A sequence generates a vesicle binding protein.

The pRIT2T-Sy3 gene product was phosphorylated in uitro using the catalytic subunit of CAMP-dependent protein kinase to a calculated stoichiometry of 1.1 mol of phosphate/m01 of protein and tested for its ability to interact with synaptic vesicles. The gene product of pRIT2T-Sy3 is the shortest fusion protein which contains both the phosphorylation site and a vesicle binding domain. There was no difference in

binding of the dephosphoform and phosphoform of this pro- tein to synaptic vesicles (Fig. 5B).

Domain B of Synapsin II Binds to a Protein Component of the Vesicle-Trypsinization of small synaptic vesicles strongly reduced the binding of the recombinant protein A- synapsin II fusion proteins to these vesicles (Fig. 5A), sug- gesting that this interaction is mediated by a protein compo- nent on the vesicles which is sensitive to trypsin. This protein might be specific for small synaptic vesicles which would explain the fact that the gene product of pRIT2T-Syl did not bind to liver microsomes or red blood cell membranes. Fig. 6 shows the protein staining pattern of synaptic vesicles before (lanes 1, 3, and 5) and after (lanes 2, 4, and 6) trypsinization. The silver-stained gel (lanes 1 and 2) demonstrates that under the conditions employed, trypsin treatment did not cause a major change in the protein composition. In particular, it can be seen that synaptophysin, a major integral membrane pro- tein of synaptic vesicles, was virtually unaffected by the protease treatment. This was confirmed by the immunoblot probed with a polyclonal antibody against synaptophysin (lanes 3 and 4). In contrast, ~65 was degraded under these circumstances, as seen in the silver-stained gel and an im- munoblot (lanes 5 and 6), developed with a monoclonal anti- body against ~65. These results support ~65 as a candidate

FIG. 6. Limited trypsinization of synaptic vesicles. Purified stripped synaptic vesicles were trypsinized as described under “Ex- perimental Procedures.” 5 rg each of intact (lanes I, 3, and 5) or trypsinized synaptic vesicles (lanes 2, 4, and 6) were separated on a 7.5-15s gradient SDS-gel and stained with silver (lanes 1 and 2). A portion of the gel was blotted onto nitrocellulose filter and probed with a polyclonal antibody against synaptophysin (lanes 3 and 4) and a monoclonal antibody against p65 (lanes 5 and 6).

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for a synapsin II binding protein. Finally, an analysis, using high performance liquid chromatography and gas-phase se- quencing, of the peptides cleaved off by the trypsin treatment from the synaptic vesicles revealed that synaptobrevin is also very rapidly degraded. Thus, from 20 peptides analyzed, 15 contained synaptobrevin sequences.’

DISCUSSION

Small synaptic vesicles are highly specialized secretory organelles that have a central function in the packaging, storage and release of neurotransmitter. The proteins specif- ically associated with the synaptic vesicle membrane are likely to participate in distinct aspects of the exocytotic and endo- cytotic process, although the molecular mechanisms are not yet understood. So far only a small number of synaptic vesicle proteins have been analyzed in mammalian brain: synapsins I and II (for review, see DeCamilli and Greengard, 1986; DeCamilli et al., 1991), synaptophysin (Jahn et al., 1985; Wiedenmann and Franke, 1985; Sudhof et al., 1987; Leube et al., 1987; Buckley et al., 1987), synaptobrevin/VAMP-1 (Trimble et al., 1988; Baumert et al., 1989; Siidhof et al., 1989a; Elferink et al., 1989), p65 (Matthew et al., 1981), and SV2 (Buckley and Kelly, 1985).

The best characterized of these proteins from a functional point of view are the synapsins, a family of neuronal phos- phoproteins. It has been suggested that synapsin I may act as a link between the cytoskeletal matrix and the vesicle surface (reviewed in DeCamilli and Greengard, 1986; Linsted and Kelly, 1987; Trimble and Scheller, 1988; DeCamilli et al., 1991). Synapsin I may regulate the number of vesicles avail- able for release by sequestering them to the cytoskeleton and thereby preventing their fusion with the presynaptic mem- brane. Thus, an understanding of the interaction of synapsin with synaptic vesicles is important in defining the function of synapsin in the nerve terminal.

The finding that phosphorylation of synapsin I by Ca’+/ calmodulin-dependent protein kinase II decreases the affinity of synapsin I for the vesicle membrane generated a model in which the basic tail of synapsin I (containing the two Ca2+/ calmodulin-dependent protein kinase II phosphorylation sites) is in contact with the vesicle membrane while the globular head interacts with the cytoskeleton (DeCamilli and Greengard, 1986; Trimble and Scheller, 1988; Hirokawa et al., 1989). Two recent experimental results have led to an expan- sion of this model.

Cloning of the cDNAs of the four synapsins (Sudhof et al., 1989b) demonstrated that synapsin II has a high degree of homology with synapsin I in the NH&erminal domains. Synapsin II binds to synaptic vesicles although it lacks the basic tail of synapsin I and thus phosphorylation sites 2 and 3 for Ca’+/calmodulin-dependent protein kinase II. Therefore synapsin II must contain binding site(s) in the region corre- sponding to the NHp-terminal domain of synapsin I. The data presented in this paper provide evidence that synapsin II binds at domain B to a protein component in the synaptic vesicle membrane.

Structure-function studies of the binding of synapsin I have recently been carried out (Benfenati et al., 1989a, 1989b) using fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid (Bahler et al., 1989). This reagent produced a 29-kDa NHz-fragment of synapsin I which bound to phospholipid vesicles and to the phospholipid bilayer of synaptic vesicles. From the deduced sequence of the synapsin family, it was suggested that the central homologous

2 G. Thiel, C. A. Slaughter, P. Greengard, and T. C. Siidhof, unpublished observations.

region of synapsins I and II (domain C) might be responsible for the interaction with lipids. Benfenati et al. (1989a) also showed that the 35/36-kDa and 39/40-kDa COOH-terminal fragments of synapsin I also contained a binding site. This binding was reduced or abolished by exposure of vesicles to trypsin, indicating an association with a protein component of the vesicle membrane. These studies led to a model of synapsin I in which the head domain binds to the phospholipid bilayer and to F-actin and the tail domain interacts with a synaptic vesicle membrane protein (Bahler et al., 1989; Ben- fenati et al., 1989a, 198913).

Based on the present study we further expand this model. The results presented in this paper indicate that domain B of synapsin II is involved in binding to a protein component of small synaptic vesicles. Domain A does not participate in binding to vesicles and no function has been established so far, even though it contains the phosphorylation site for CAMP-dependent protein kinase and Ca*+/calmodulin-de- pendent protein kinase I. Recent genomic cloning of synapsin I (Siidhof, 1990) revealed that domain A/domain B is encoded by a single exon, while the central homologous domain C is broken up into 10 exons. Our results therefore indicate that exon 1 encodes a binding domain for synaptic vesicles. We mapped this domain between amino acids 43 and 121 of synapsin II. The gene products of pRIT2T-Sy3 and pRITBT- Sy5 both bind to synaptic vesicles, although they do not contain overlapping synapsin II sequences. Synapsin II might contain two distinct vesicle binding sites in domain B, mapped between residues 43-14 and 75-121, respectively. Alterna- tively, these sites might be parts of a single binding domain.

The synapsins also contain at least one phospholipid bind- ing site, which has been attributed to domain C. Unfortu- nately, we could not investigate the binding behavior of do- main C, the central homologous domain, in more detail be- cause recombinant proteins containing only this region were completely insoluble even when fused to a truncated protein A. In general the hydrophobicity of recombinant proteins (discussed by Marston, 1986) is the major disadvantage of this approach. At higher concentrations, the protein A-syn- apsin II fusion proteins aggregated and generated high back- ground values (above 50%) in the in vitro vesicle binding assay. Therefore, we did not attempt to calculate Kd values.

In conclusion, we were able to demonstrate the specificity of the binding site in domain B in two different ways.

1. Several recombinant proteins did not bind to vesicles. The fusion proteins containing the 42 NH*-terminal amino acids of synapsin II (encoded by pRIT2T-Sy4) or the 57 and 21 carboxyl-terminal residues of synapsin Ia and synapsin Ib, respectively, did not bind to small synaptic vesicles, suggesting that both the amino terminus of the synapsins and the differ- entially spliced carboxy termini of synapsin I must have functions other than anchoring synapsin to the vesicle. More- over the truncated protein A and the protein A-synaptophysin fusion protein did not bind to synaptic vesicles. However, by fusion of 32 residues (pRIT2TSy3) or 47 residues (pRITZT- Sy5) of synapsin II, that included portions of the B domain, to the truncated protein A, we generated a protein which showed reproducible cosedimentation with synaptic vesicles.

2. The experiments with trypsinized synaptic vesicles and liver microsomes showed that the recombinant protein A- synapsin II fusion proteins required the intact synaptic vesicle membrane for binding. These results indicate that the ob- served binding behavior is specific for small synaptic vesicles and is not a nonspecific interaction between a hydrophobic protein and any membrane.

The data presented here indicate that domain B of synapsin

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II interacts with a protein component on the vesicle mem- brane. So far, attempts to identify this protein using affinity columns or cross-linking reagents have been unsuccessful (data not shown). Synaptophysin was virtually unaffected by trypsin treatment under the conditions employed, making it unlikely that this protein is involved in the binding process. It will be of interest to evaluate the possibility that synapto- brevin or p65 can bind synapsin. It seems possible that synapsin I and synapsin II interact with more than one vesicle component making it difficult to elucidate the nature of the binding.

Finally, all of the binding sites for the synapsins have been mapped in vitro, using purified stripped synaptic vesicles as receptor preparation, and purified synapsin I, fragments of synapsin I, or recombinant synapsin constructs as ligands. These binding sites include the hydrophobic binding site located in the head domain of synapsin I (Benfenati et al., 1989a, 1989b), the protein binding site in the tail region of synapsin I (Benfenati et al., 1989a) and the protein binding site in domain B of synapsin II (present study). In addition, binding of synapsin I to F-actin has been demonstrated in vitro (Bihler and Greengard, 1987; Bahler et al., 1989; Pe- trucci and Morrow, 1987). It will be important to attempt to confirm these data in vivo, using transfection techniques.

Acknowledgments-We wish to thank all members of the Green- gard and Sudhof laboratories for their contributions (antibodies, purified CAMP-dependent protein kinase and purified rat synapsin II, plasmid pRIT2T-38e). We also thank L. Reichard, University of California, San Francisco, for providing anti-p65 antibodies. We thank Martin Bahler, Fabio Benfenati, Piera Cicchetti, and Andrew J. Czernik for critical reading of the manuscript.

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Synapsin II. Mapping of a domain in the NH2-terminal region which binds to

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