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Originally published In Press as doi:10.1074/jbc.M910092199 on April 11, 2000

J. Biol. Chem., Vol. 275, Issue 23, 17481-17487, June 9, 2000
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A Conserved Membrane-spanning Amino Acid Motif Drives Homomeric and Supports Heteromeric Assembly of Presynaptic SNARE Proteins*

Rico LaageDagger , Jan Rohde, Bettina Brosig, and Dieter Langosch§

From the Department of Neurobiology, Universität Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany

Received for publication, December 15, 1999, and in revised form, March 20, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha -helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular membrane fusion events, e.g. constitutive organelle traffic or Ca2+-regulated neurotransmitter release, require conserved sets of membrane proteins, designated SNAREs.1 The best characterized SNAREs are those mediating exocytosis of synaptic vesicles in neurons (reviewed in Refs. 1-4). In detergent extracts from presynaptic nerve terminals, the single-span integral membrane SNAREs synaptobrevin (also referred to as VAMP) and syntaxin together with the peripheral membrane SNARE protein SNAP-25 form a stable ternary complex that is disassembled in vitro after binding of soluble alpha -SNAP by the ATPase NSF (5, 6). According to the original SNARE hypothesis (7), interaction between these SNARE partners bridges opposing vesicular and plasma membranes. Therefore, assembly and disassembly of ternary SNARE complexes would proceed in a trans-configuration that is regarded essential for vesicle docking, priming and/or fusion (8-12). On the other hand, SNARE complexes are also found on the vesicular (13, 14) as well as the plasma (15) membrane in a cis-configuration, i.e. side by side. Protein domains involved in the binary and ternary interactions leading to SNARE complex formation have been originally identified by in vitro binding studies using recombinant soluble fragments of synaptobrevin II, syntaxin 1A, and SNAP-25 as follows: (i) the cytoplasmic domain of synaptobrevin II; (ii) a carboxyl-terminal, membrane-proximal region of syntaxin 1A; and (iii) carboxyl-terminal plus amino-terminal regions of SNAP-25 (16-21). More recently, structural studies confirmed that previously predicted cytoplasmic coiled coil domains of synaptobrevin II (H1, H2) and syntaxin 1A (H3) form a tightly packed parallel four-helical bundle together with two helices derived from SNAP-25 (HA and HB) in the SNARE complex (22, 23). In contrast, the role of the carboxyl-terminal TMSs located at one side of the SNARE complex (24-26) in these interactions has only been characterized in part.

Transmembrane domains are known to participate in oligomerization of many different integral membrane proteins. It is thought that TMS self-assembly is driven by a close packing of their characteristically shaped surfaces that are defined by their primary structures (reviewed in Ref. 27). We have previously shown that the homodimeric structure originally observed for native synaptobrevin (28-30) is preserved with recombinant synaptobrevin II, where it depends on a specific amino acid motif within the TMS (31). Furthermore, a direct interaction of syntaxin and synaptobrevin TMSs in synthetic proteoliposomes was recently reported (32). Surprisingly, we found that the synaptobrevin II homodimerization motif is conserved within the TMS of syntaxin 1A. This predicted the involvement of this motif in a homotypic interaction of syntaxin 1A as well as in its heterophilic binding to synaptobrevin II.

Here, we (i) identify the minimal TMS amino acid motif required for synaptobrevin II homodimerization, (ii) show that a similar motif mediates syntaxin 1A homodimerization, and (iii) demonstrate that TMSs and cytoplasmic coiled coil domains cooperate in synaptobrevin/syntaxin heterodimerization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructs-- Rat syntaxin 1A cDNA was amplified by polymerase chain reaction with VENT polymerase (Biolabs) from a plasmid template as described previously for rat synaptobrevin II (31) with primers containing NheI (sense primer) and BglII (antisense primer) restriction sites. The amplified fragment was cut with NheI and BglII and ligated into the pET 21d (Novagen)-based plasmid pSNiR, previously cut with NheI and BamHI. In the resulting constructs, the amino termini of synaptobrevin or syntaxin are fused in frame to the carboxyl terminus of a tripartite fusion moiety consisting of the coding sequence of the HA marker epitope in case of synaptobrevin or the c-myc epitope for syntaxin, Staphylococcus aureus nuclease A, and a linker-region coding for 9 amino acids.

Construction of plasmid pToxR-A16 was described previously (33). All other pToxR constructs were made by ligating synthetic oligonucleotide cassettes encoding the desired sequences into plasmid pHKToxR(TMIl4)MalE (34) previously cut with NheI and BamHI. All constructs were verified by dideoxy sequencing.

Site-directed Mutagenesis-- All mutants were made by oligonucleotide-directed mutagenesis performed according to Kunkel et al. (35) on single-stranded templates (T7 Mutagene-kit, Bio-Rad). All mutations were verified by dideoxy sequencing.

ToxR Activity Assays-- ToxR activities were determined in quadruplicates in 3-11 independent experiments as described (33) and are given in Miller units (mean ± S.E.). Western blot analysis was done as described (33). PD-28 cell growth assays were done as described (36), and the absorbance was read at 650 nm at a pathlength of 6 mm. To correct for slightly different membrane insertion efficiencies, the beta -galactosidase activities elicited by the mutant sequences were normalized to the A650 values obtained after 48 h of cell growth.

Expression and Radiolabeling of Recombinant Proteins-- Proteins, encoded by pSNiR vectors, were expressed in Escherichia coli BL21(DE3)pLsyS (Novagen) as described (31), solubilized with 2% (v/v) polyoxyethylene 9 lauryl ether (Sigma) for mild SDS-PAGE, or with 2% (w/v) CHAPS (Applichem, Darmstadt) in solubilization buffer (50 mM Hepes, pH 7.9, 1 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) for all other applications. The usual concentration of recombinant protein after solubilization was ~1 mg/ml. Biosynthetic labeling was performed by addition of 7.5 µCi of [35S]methionine/cysteine (2:1) (Amersham Pharmacia Biotech) per ml of bacterial culture 15 min after induction of expression with isopropyl-beta -D-thiogalactopyranoside.

Cross-linking-- CHAPS-solubilized proteins (see above) were treated with DSS (Pierce) dissolved in dimethyl sulfoxide. Dimethyl sulfoxide never exceeded 2% (v/v) of the reaction volume. The samples were shaken for 5 min at room temperature, and reactions were then quenched with 100 mM Tris, pH 7.4, for 10 min. The samples were precipitated with methanol (37) prior to SDS-PAGE.

Overlay Blotting-- CHAPS-solubilized proteins were separated by SDS-PAGE (~20 µg/lane) and subsequently blotted onto nitrocellulose membrane (Amersham Pharmacia Biotech Hybond-C pure). The membrane was blocked for 30 min in TBB (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% (v/v) Triton X-100, 1 mM EDTA, 4% (w/v) nonfat dry milk powder). The blocking solution was replaced by 15 µg/ml 35S-labeled wt protein of the cognate SNARE partner in TBB while shaking was continued for 2 h at room temperature. The blots were washed twice for 10 min in TBS (50 mM Tris-Cl, pH 8.0, 150 mM NaCl) and once in TWB (TBS + 0.1% (v/v) Triton X-100). Blots were air-dried and exposed to a BAS-MP imaging plate (Fuji, Japan) overnight, and bound 35S-labeled protein was quantified using a BAS-1000 bio-imaging analyzer (Fuji, Japan).

Co-immunoprecipitation-- CHAPS-solubilized HA-tagged synaptobrevin II derivatives were incubated with wt 35S-labeled syntaxin (final concentration 250 ng/µl) in solubilization buffer at 4 °C for 2 h. The monoclonal anti-HA antibody 12CA5 (a kind gift of Dr. Lerner, Scripps Institute, San Diego, CA) was added at 20 ng/µl, and incubation was continued with shaking overnight at 4 °C. Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) was added, pelleted (Hereaus Biofuge 4000 rpm) after 2 h, washed twice with buffer A (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2% (v/v) Triton X-100), twice with buffer B (10 mM Tris-Cl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.2% (v/v) Triton X-100), and once with buffer C (10 mM Tris-Cl, pH 7.5, 0.2% (v/v) Triton X-100). After washing, bound proteins were eluted with SDS sample buffer and separated by SDS-PAGE. Gels were dried and exposed to a BAS-MP imaging plate (Fuji, Japan), and the amount of co-precipitated radiolabeled syntaxin was quantified using a BAS-1000 bio-imaging analyzer (Fuji, Japan).

Mild SDS-PAGE and Western Blotting-- Proteins solubilized with 2% polyoxyethylene 9 lauryl ether were precipitated with methanol (37), redissolved in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, 6 M urea, 50 mM dithiothreitol, 20% (w/v) sucrose), separated by SDS-PAGE (5 µg/lane), and visualized by Western blotting using the HA-mAb 12CA5 for HA-tagged synaptobrevin or the myc-mAb 9E10 for myc-tagged syntaxin 1A as described (31). Minigels were run at 4 °C, and samples were not boiled prior to electrophoresis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Minimal Homodimerization Motifs of Synaptobrevin II and Syntaxin 1A-- We previously described an amino acid motif in the synaptobrevin II TMS that is essential for its homodimerization. Mutational analysis had identified 6 amino acid residues (Leu-99, Ile-102, Cys-103, Leu-107, Ile-110, and Ile-111) whose exchange to alanine significantly reduced self-interaction of the recombinant proteins expressed in E. coli as analyzed by SDS-PAGE under mild conditions. Simultaneous mutation of three of these residues (L99A/C103A/I111A = syb-mult) abrogated homodimerization almost completely (31) (Figs. 1A and 2A). To examine whether this motif is sufficient for synaptobrevin II TMS-TMS interaction, we compared a set of synaptobrevin II mutants for self-interaction by mild SDS-PAGE. Replacing the TMS by an oligoalanine sequence (syb-A15), which does not self-interact (33), reduced homodimerization as efficiently as the previously characterized "mult" mutations (Fig. 2A). Grafting the critical 6 residues onto this oligoalanine background (syb-A8) completely restored homodimerization thus identifying them as minimal amino acid motifs responsible for synaptobrevin II TMS-TMS interaction. As mutation of alanine 104 previously did not influence homodimerization (31), this residue is not considered as part of this motif.


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  		Fig. 1.   Amino acid sequences of synaptobrevin II and syntaxin 1A TMSs and neighboring cytoplasmic coiled coil domains. A, an alignment of the TMSs reveals conservation of the 6 residues previously found important for synaptobrevin II homodimerization in syntaxin 1A (boxed). B, TMS mutants where different combinations of the central 15 amino acids were mutated to alanine (in boldface). Positions corresponding to the minimal 6- or 8-residue dimerization motifs identified in this study are boxed (see text). Syx-cyt was made by deletion of the syntaxin 1A TMS starting from Lys-265. C, the TMS proximal cytoplasmic domains are characterized by specific heptad repeat patterns of hydrophobic amino acids (indicated by a and d). In syb-60/84 and syx-230/251, five of these amino acids were exchanged to alanine (in boldface).


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  		Fig. 2.   Homodimerization of synaptobrevin II and syntaxin 1A. Wild type and mutated full-length proteins were separated by SDS-PAGE and visualized by Western blotting. A, the 66-kDa homodimer of syb-wt (synaptobrevin/nuclease A fusion = 33-kDa monomer) is disrupted upon different TMS mutations (syb-A15 and syb-mult) but preserved with a 6-residue TMS motif (syb-A8). B, the 100-kDa homodimer of syx-wt (syntaxin/nuclease A fusion = 50-kDa monomer) is disrupted upon TMS-deletion (syx-cyt) or mutation (syx-A15 and syx-mult) but appears even stronger with a conserved 6-residue TMS motif (syx-A8).

A sequence comparison identified 5 (Ile-270, Cys-271, Leu-275, Ile-278, and Ile-279) out of the 6 positions to be conserved in the TMS of syntaxin 1A, the natural binding partner of synaptobrevin II (Fig. 1A). Therefore, we examined the potential role of this motif in syntaxin 1A homodimerization. Analysis of recombinant syntaxin 1A by SDS-PAGE under mild conditions indeed revealed its partial homodimerization. Dimer formation was almost completely abrogated when the TMS had been deleted (syx-cyt); its central 15 hydrophobic amino acids were replaced by the oligoalanine sequence (syx-A15) or mutated in three positions (syx-mult = M267A/C271A/I279A). Grafting the motif homologous to the synaptobrevin II homodimerization motif (Met-267, Ile-270, Cys-271, Leu-275, Ile-278, and Ile-279) onto the oligoalanine sequence (syx-A8) resulted in syntaxin homodimerization that was even stronger than that of the wt protein (Fig. 2B). Thus, syntaxin 1A also forms homodimers by sequence-specific self-interaction of its TMS based on an amino acid motif almost identical to that previously identified in synaptobrevin II.

To examine whether the cytoplasmic coiled coil domains of synaptobrevin and syntaxin are involved in homodimerization, multiple mutations were made in positions relevant for binary and ternary SNARE protein interactions (Fig. 1C) (Refs. 18, 19, 23, and see below). These mutations had no detectable effect on homodimerization of both proteins, indicating that the cytoplasmic coiled coil domains do not self-interact (results not shown, but see Fig. 4).

Self-interaction of SNARE TMSs in Membranes-- To examine self-interaction of the SNARE TMSs in the absence of the cytoplasmic domains and incorporated into membranes, we used the ToxR transcription activator system. We previously established this system as a sensitive tool to study TMS-TMS interactions using the structurally well characterized glycophorin A TMS dimer for reference (36, 38). The ToxR protein is anchored by a single TMS of choice within the inner membrane of expressing E. coli cells where it is thought to exist in a monomer/dimer equilibrium. The dimeric form binds to the cholera toxin promoter thus activating expression of a downstream lacZ gene in a reporter strain (Fig. 3A). beta -Galactosidase expression is therefore diagnostic of ToxR self-assembly in the membrane.


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  		Fig. 3.   Self-assembly of SNARE TMSs in membranes as probed with the ToxR system. A, functional organization of ToxR chimeric proteins. The cytoplasmic ToxR domain is linked via a TMS of choice to the periplasmic MalE moiety. Upon dimerization, ToxR binds to the ctx promoter thus initiating lacZ transcription in the indicator cells. OM, outer membrane; IM, inner membrane. B, different levels of beta -galactosidase (beta -gal) activity reveal self-assembly of wt TMSs (syb-wt and syx-wt) which is decreased by the mult mutations but preserved with motifs of 8 amino acids (syb-A6 and syx-A6). Significant differences (p < 0.05) are marked with * and highly significant differences (p < 0.001) with ** (Student's t test). The TMS sequences expressed within the context of ToxR proteins correspond to those given in Fig. 1B. The activity elicited by the oligoalanine sequence (A16) reflects the background signal of the system (marked with a dashed line). C, the expression levels of the ToxR proteins with the SNARE TMSs in FHK12 cells were similar, whereas the A16 construct was overexpressed (33) as revealed by Western blotting. D, all constructs, except the negative control with the deleted TMS (Delta TM), supported the growth of MalE-deficient PD28 cells to similar degrees indicating their similarly efficient membrane integration. MU, Miller units.

Here, we replaced the ToxR TMS by the synaptobrevin II or syntaxin 1A TMSs to study their self-interaction. The transcriptional activities of these chimeric ToxR proteins indicated that both SNARE TMSs self-interacted similarly well in the membrane. The degrees of interaction were comparable to that of a previously characterized membrane-spanning leucine zipper (33) and below that of the glycophorin A TMS (36, 38). Since the signals were reduced by the mult mutations to statistically significant degrees (Fig. 3B; two-tailed Student's t test, p < 0.05), the interactions are sequence-specific and involve the same faces of the transmembrane helices as determined for full-length proteins in detergent solution. The weaker effects of the mult mutations found here as compared with detergent solution is assumed to result from higher protein concentrations and/or preorientation of the interacting domains in the membrane (39). The minimal interaction motifs were determined using an oligoalanine host sequence (A16) previously shown to partially partition into the membrane where it stays largely monomeric (33). The 6-residue motifs, which were sufficient for homodimerization of full-length SNAREs in detergent (Fig. 2), partially restored self-interaction in the membrane. To obtain wild-type levels of homodimerization, however, these motifs had to be expanded by two additional conserved isoleucine residues completing the contiguous areas of interfacial residues (Figs. 3B and 7). To exclude that different concentrations of the ToxR chimeric proteins in the membranes distorted the signals, we ascertained their similar expression levels by Western blot analysis (Fig. 3C). Furthermore, we assessed the efficiency of the ToxR constructs to integrate into the inner bacterial membrane by testing their ability to functionally complement the maltose-binding protein (MalE) deficiency of PD28 cells. Due to a deletion in MalE, this strain is unable to grow in minimal medium with maltose as the only carbon source (40). In cells expressing correctly inserted ToxR membrane proteins, however, the MalE domain allows maltose uptake and thus cell growth (36). All constructs complemented MalE deficiency to comparable degrees, thus confirming their similarly efficient membrane integration; a ToxR protein with deleted TMS (Delta TM) did not support cell growth (Fig. 3D) as expected (36).

In sum, both SNARE TMSs are capable of self-assembling in membranes in the absence of the cytoplasmic domains. Conserved motifs of 8 amino acids are sufficient to mimic these homotypic interactions.

Heterodimerization of Synaptobrevin II and Syntaxin 1A-- Conservation of the self-interacting TMS motifs suggested that TMS-TMS interactions may also be important for heterodimerization of both proteins. This is in line with a recently reported contribution of the TMSs to synaptobrevin/syntaxin interaction (32). On the other hand, synaptobrevin II and syntaxin 1A are known to interact via cytoplasmic coiled coil domains in binary as well as in ternary complexes including SNAP-25 (16, 18-20, 22, 23). The hallmark of coiled coil structures is that the a and d positions within repeated abcdefg motifs form the hydrophobic core of the helix-helix interfaces. Mutating a and d positions of the H3 region of syntaxin previously resulted in loss of ternary as well as binary interactions (18).

To compare the contribution of the cytoplasmic coiled coil domains and the TMSs to heterodimerization, we generated mutations in either part of synaptobrevin II and syntaxin 1A. To test these proteins (tagged with HA or myc epitopes, respectively) for their ability to form homo- and heterodimers, they were co-incubated, cross-linked with DSS, and analyzed by SDS-PAGE followed by immunoblotting. Wild-type proteins formed homodimers plus an additional band of intermediate apparent molecular weight that was detected with both antibodies and thus identified as the heterodimer. This result suggests that homodimerization competes with heterodimerization. Mutating five a and d positions in the coiled coil domains of synaptobrevin (syb-60/84) or syntaxin (syx-230/251) (Figs. 1C and 7) strongly reduced heterodimerization, as expected (Fig. 4). In comparison, the TMS mult mutations had a detectable but somewhat less pronounced effect on heterodimerization as assayed by DSS cross-linking or SDS-PAGE analysis under mild conditions (data not shown).


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  		Fig. 4.   Cross-linking of synaptobrevin II/syntaxin 1A heterodimers. Left panel, Western blot with the myc-mAb (staining the syntaxin fusion protein = syx) reveals the 50-kDa syntaxin monomer, the 100-kDa syntaxin homodimer (hom), and the 82-kDa synaptobrevin/syntaxin heterodimer (het) upon cross-linking with 150 µM DSS (1st lane). Right panel, Western blot with the HA-mAb (recognizing the synaptobrevin fusion protein = syb) reveals the 33-kDa synaptobrevin monomer, the 66-kDa synaptobrevin homodimer (hom), and the 82-kDa heterodimer (het) upon cross-linking (4th lane). Point mutations in the coiled coil domain of either synaptobrevin (syb-60/84) or syntaxin (syx-230/251) strongly reduced heterodimer formation (2nd, 3rd, 5th, and 6th lanes), without affecting homodimerization. (Note that the apparent molecular weight of syb-60/84 is slightly larger than that of syb-wt.)

To evaluate quantitatively the roles of coiled coil domains and TMSs in heterophilic interaction, we developed an overlay assay. Equal amounts of wt and mutant proteins were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with 35S-labeled wt-binding partners. By quantifying bound radioactivity, different degrees of heterodimerization were determined for wt and mutants. The results confirmed the importance of both coiled coil domains for heterophilic binding (Fig. 5A). Importantly, the multiple TMS mutations (syb-mult and syx-mult) or replacement of the TMSs by the oligoalanine sequence (syb-A15 and syx-A15) also significantly decreased binding to their respective wt SNARE partner (Fig. 5). To identify the minimal amino acid motifs responsible for heterophilic TMS-TMS interaction, we tested the proteins with the minimal homodimerization motifs. In case of syntaxin 1A, the motif of 6 residues (syx-A8) was sufficient for wt level of heterodimerization with wt synaptobrevin II, whereas the 8-residue motif that completely restored homodimerization in the ToxR system (syb-A6) was required in the reverse configuration.


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  		Fig. 5.   Heterodimerization of synaptobrevin II and syntaxin 1A analyzed by an overlay assay where unlabeled proteins were electrophoresed, blotted, and subsequently probed with the cognate 35S-labeled wt SNARE. A, left panel, wt 35S-synaptobrevin bound less efficiently to syntaxin mutated within the cytoplasmic domain or the TMS as compared with the wild-type (= 100%) or the protein with the 6 amino acid TMS interaction motif. Right panel, wt 35S-syntaxin bound less efficiently to synaptobrevin mutated within the cytoplasmic domain or the TMS as compared with the wild type (= 100%) or the protein with the 8-amino acid TMS interaction motif. The bars represent mean values from several independent experiments (n = 6-16 ± S.E.; *, p < 0.05; **, p < 0.001). See Fig. 1 for the sequences. B, representative autoradiograms. C, Coomassie-stained gels reveal that equal amounts of proteins were blotted. The order of samples in B and C follows that in A.

By using an independent experimental approach, we investigated the influence of the mutations on the ability of synaptobrevin to co-precipitate 35S-labeled wt syntaxin from detergent solution. Upon co-incubation, we immunoprecipitated the synaptobrevin proteins and quantitated co-precipitated syntaxin upon SDS-PAGE. In agreement with the overlay assay (Fig. 5), the coiled coil mutant syb-60/84 as well as the TMS mutants syb-mult and syb-A15 co-precipitated significantly less syntaxin compared with wt synaptobrevin, whereas syb-A6 with the 8-residue TMS motif was as efficient as the wt protein (Fig. 6).


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  		Fig. 6.   Heterodimerization of synaptobrevin II and syntaxin 1A tested by co-immunoprecipitation. HA-tagged wt and mutated synaptobrevin proteins were co-incubated with 35S-labeled wt syntaxin 1A and immunoprecipitated with the HA-mAb. Precipitates were separated by SDS-PAGE, and co-precipitated 35S-syntaxin was quantified. A, relative binding intensity of wt syntaxin 1A to different synaptobrevin II proteins. The co-precipitated amounts of 35S-syntaxin were strongly reduced (*, p < 0.05; **, p < 0.001) with the cytoplasmic or TMS mutants relative to the wt protein (=100%) or the protein with the 8-amino acid TMS interaction motif. See Fig. 1 for the sequences. The bars represent mean values of at least 5 independent experiments ± S.E. B, typical autoradiogram of precipitated 35S-syntaxin. The order of samples in B follows that in A.

Together, these findings show that heterophilic interaction of synaptobrevin II with syntaxin 1A is not only dependent on soluble coiled coil structures but also on specific interactions of their TMSs. Conservation of the minimal TMS motifs mediat-ing homo- and heterodimerization indicates that both types of interaction involve the same faces of the alpha -helical interaction domains.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our data reveal that conserved amino acid motifs within the TMSs are crucial for homo- and heterodimerization of synaptobrevin II and syntaxin 1A, two natural binding partners in the presynaptic nerve terminal. Previously, we had shown that homodimerization of synaptobrevin II, originally observed for the native protein (28-30), depends on a specific amino acid motif in its TMS (31). Interestingly, this motif is almost completely conserved in the syntaxin 1A TMS. In analogy to synaptobrevin II, we show here that mutating or deleting the TMS nearly abolished homodimerization of syntaxin 1A. Furthermore, within full-length proteins, sets of 6 residues form the minimal TMS dimerization motifs when grafted onto an oligoalanine host sequence. By using the ToxR system, we demonstrate that both TMSs self-assemble even in the absence of the cytoplasmic domains in a membrane environment. Here, the amino acid motifs sufficient for wild-type level homodimerization contain two additional conserved isoleucine residues that complete contiguous interfacial areas modeled onto alpha -helical surfaces (Fig. 7).


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  		Fig. 7.   Amino acid motifs driving interaction of cytoplasmic and TMS helices. Helical net representations are given for synaptobrevin II from position 59 and for syntaxin 1A from position 229. Residues at a and d positions at the core of the cytoplasmic coiled coil domains are hatched. Those residues within the cytoplasmic domains or the TMSs whose mutation affected homo- and/or heterodimerization in this study are shaded. The minimal 6-residue motifs within the TMSs, which were required for homodimerization of full-length proteins, are highlighted by dark shading. Both additional amino acids required for homodimerization in membranes or heterodimerization of synaptobrevin with syntaxin are marked by lighter shading. See text for details.

To examine whether the interaction motifs identified in rat synaptobrevin II and syntaxin 1A are conserved in other SNARE isoforms, we searched several protein sequence data bases (Swiss-Prot, PIR, and TREMBL) with the synaptobrevin II motif ILXXICXXILXXII where X represents any amino acid except arginine, lysine, glutamate, histidine, aspartate, and proline in order to retrieve selectively unkinked TMSs. We allowed for up to two mismatches to detect closely related motifs. The search identified human, rat, mouse, and bovine synaptobrevin II (0 mismatches) and Xenopus laevis synaptobrevin II, rat synaptobrevin IIB, synaptobrevins from zebrafish and japanese pufferfish, mouse syntaxin 1A (1 mismatch), rat endobrevin, human syntaxin 1A, and mouse syntaxin 3B and 3C (2 mismatches). Thus, it appears that the rat synaptobrevinII/syntaxin 1A motif is partially conserved in other SNARE protein family members, especially in orthologs.

Homodimerization of the TMS motifs reflects their spatial self-complementarity that allows tight packing of amino acid side chains. The near identity of the motifs in synaptobrevin II and syntaxin 1A therefore predicted that the TMSs also support heterophilic interaction of both proteins. Our data confirm this prediction since heterodimerization competes with homodimerization and is also reduced by mutating either TMS. Furthermore, the minimal homodimerization motifs suffice for a wild-type level of heterophilic interaction. Assuming alpha -helicity of both TMSs, these minimal motifs wrap around their surfaces in a right-handed fashion (Fig. 7). This suggests that the helices are tilted relative to each other in the dimer with negative packing angles in order to maximize side chain packing which is reminiscent of the glycophorin A transmembrane helix-helix pair (41).

In agreement with previous data (18, 19, 22, 23), our present results show that mutating a and d positions within the coiled coil domains also reduced heterodimerization without, however, affecting homodimerization. Thus TMS-TMS interactions appear as the main driving forces for homodimerization, whereas heterodimerization requires two distinct assembly domains, the cytoplasmic coiled coils and the TMSs.

Various lines of previous evidence indicate that both domains are important for the function of SNARE proteins. Caenorhabditis elegans mutants with impaired neurotransmission contained synaptobrevin (snb-1) or syntaxin (unc64) homologs with point mutations at a or d positions in the coiled coil region, a frameshift midway in the snb-1 TMS (42), or an unc64 TMS truncated to 16 hydrophobic residues (43). For both TMS mutants, the residual hydrophobic domains should be sufficient to function as membrane anchors (44), which is supported by the apparent localization of the snb1 mutant to synaptic vesicles (43). The notion that the function of SNARE TMSs extends beyond their role as membrane anchors is underscored by the existence of syntaxin isoforms in C. elegans (43) and vertebrates (45, 46) distinguished only by their TMSs. Furthermore, alternatively spliced variants of synaptobrevin I differing only in their TMSs have been reported to localize to distinct subcellular compartments (47). It is therefore tempting to speculate that the structures of, and presumably interactions between, SNARE protein TMSs are important for their function.

TMS-TMS interactions may play a role at different stages of SNARE protein function. First, heterophilic TMS-TMS interaction may stabilize the SNARE complex. The original SNARE hypothesis postulated that synaptobrevin as a v-SNARE of synaptic vesicles interacts with the t-SNAREs syntaxin and SNAP-25 of the plasma membrane in a trans-configuration (5-7). On the other hand, ternary SNARE complexes are also present in synaptic vesicles (13, 48, 49) and the plasma membrane (15) in a cis-configuration allowing for TMS-TMS interactions. Indeed, the presence of the TMSs increased the thermal stability of a trypsin-resistant core SNARE complex (20). Furthermore, Margittai et al. (32) reported increased stability of the SNARE complex including the TMSs against disassembly mediated by alpha -SNAP and NSF as well as the appearance of the syb/syx-heterodimer upon partial disassembly. Evidence that co-localization of v- and t-SNAREs in the same membrane might be of functional relevance comes from yeast vacuole fusion (14), where synaptobrevin and syntaxin homologs form a pentameric cis-complex in one vacuole (50).

Second, Poirier et al. (20) provided evidence for another potential role of TMSs interactions. Strong multimerization of SNARE complexes was observed with both TMSs present; it was less pronounced when the TMS of either synaptobrevin II or syntaxin 1A was deleted and absent without TMSs, suggesting that multimerization is due to inter-SNARE TMS-TMS interactions.2 In line with this, electron microscopy revealed dimers and multimers of native or recombinant SNARE complexes that associated at the sites of the TMSs; multimerization was absent with SNARE complexes composed of the cytoplasmic domains only (26). Although the function of SNARE complex multimerization is currently not clear, it may play a role in the fusion reaction. This is supported by the observation of multimeric superstructures that appear to be functional intermediates of certain fusogenic viral membrane proteins. Enveloped viruses fuse with target membranes by way of viral glycoproteins that, in analogy to SNAREs, consist of carboxyl-terminal TMSs and soluble coiled coil domains that assemble to rod-shaped trimeric helical bundles (51). Interestingly, the concerted action of at least three hemagglutinin trimers is required for fusion of influenza virus with the endosomal membrane (52). Likewise, fusion mediated by baculovirus surface glycoprotein 64 involves the assembly of transient multimeric complexes assembled by lateral association of stable glycoprotein 64 trimers (53). Upon multimerization of these fusogenic viral or SNARE protein complexes, the membrane-spanning domains may form a scaffold for fusion pores shown to precede virus-mediated bilayer fusion (54) or detected prior to catecholamine release from chromaffin granules (55), or serotonin release from leech neurons (56).

Third, SNARE TMS-TMS interactions may be directly involved in membrane fusion. Since the formation of the parallel coiled coil may proceed from the amino termini of SNARE proteins interacting in trans, the fusion of vesicular and plasma membranes may result from a successive "zippering up" of cytoplasmic domains that closely juxtaposes vesicle and target membranes (9, 22). The interaction of the TMSs may then be the final step completing membrane merger. As noted above, the arrangement of residues within the minimal TMS motifs suggests negative packing angles of the interacting alpha -helices. Since the cytoplasmic coiled coil domains assemble with positive packing angles (22), both assembly domains may be separated by a flexible linker region. In agreement with this, insertion of proline residues intended to disrupt a potential helical continuity between coiled coil domains and TMSs did not abolish SNARE protein-mediated liposome fusion (57). Thus, both interaction domains are considered to be largely independent of each other which may allow for the structural flexibility required for fusion.

    ACKNOWLEDGEMENTS

We thank Dr. C. Ungermann for critically reading the manuscript and Dr. W. B. Huttner for continuous support.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 317 and the Heisenberg Program and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Biochemie-Zentrum Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.

§ To whom correspondence should be addressed: Institut für Neurobiologie, Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Tel.: 06221-548696; Fax: 06221-544496; E-mail: Langosch@sun0.urz.uni-heidelberg.de.

Published, JBC Papers in Press, April 11, 2000, DOI 10.1074/jbc.M910092199

2 R. Laage, J. Rohde, B. Brosig, and D. Langosch, unpublished results.

    ABBREVIATIONS

The abbreviations used are: SNARE, SNAP (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein) receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; DSS, disuccinimidyl suberate; HA, hemagglutinin; MalE, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; SNAP-25, synaptosomal associated protein of 25 kDa; TMS, transmembrane segment; VAMP, vesicle associated membrane protein; wt, wild type; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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