The Transmembrane Domain of Syntaxin 1A Is Critical for Cytoplasmic Domain Protein-Protein Interactions
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m011687200
ISSN1083-351X
AutoresJessica L. Lewis, Min Dong, Cynthia A. Earles, Edwin R. Chapman,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoAssembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin protein-protein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, α/β-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a length-dependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins. Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin protein-protein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, α/β-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a length-dependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins. synaptosome-associated protein of 25 kDa soluble NSF attachment protein soluble NSF attachment protein receptor transmembrane domain polyacrylamide gel electrophoresis polymerase chain reaction glutathioneS-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Syntaxin 1A was initially identified as a 35 kDa protein in the plasma membrane of amacrine cells (1Barnstable C.J. Hofstein R. Akagawa K. Brain Res. 1985; 352: 286-290Crossref PubMed Scopus (96) Google Scholar), as a subunit of Ca2+ channels (2Inoue A. Obata K. Akagawa K. J. Biol. Chem. 1992; 267: 10613-10619Abstract Full Text PDF PubMed Google Scholar, 3Yoshida A. Oho C. Omori A. Kuwahara R. Ito T. Takahashi M. J. Biol. 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Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar) with the plasma membrane protein SNAP-251 and the synaptic vesicle protein synaptobrevin, to form a SNARE complex (9Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1614) Google Scholar). Assembly of this complex is necessary (10Littleton J.T. Chapman E.R. Kreber R. Garment M.B. Carlson S.D. Ganetzky B. Neuron. 1998; 21: 401-413Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 11Chen Y.A. Scales S.J. Patel S.M. Doung Y.C. Scheller R.H. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar) and may be sufficient to drive membrane fusion (12Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2049) Google Scholar, 13McNew J.A. Weber T. Parlati F. Johnston R.J. Melia T.J. 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Syntaxin functions as a key element in membrane traffic and membrane fusion by interacting with a wide range of other proteins. The many binding partners of syntaxin, in excess of twenty, include rbSec1A/nsec-1/munc18 (20Hata Y. Slaughter C.A. Sudhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (604) Google Scholar, 21Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (226) Google Scholar, 22Pevsner J. Hsu S.C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1445-1449Crossref PubMed Scopus (360) Google Scholar), CSP (23Wu M.N. Fergestad T. Lloyd T.E. He Y. Broadie K. Bellen H.J. Neuron. 1999; 23: 593-605Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), syntaphilin (24Lao G. Scheuss V. Gerwin C.M. Su Q. Mochida S. Rettig J. Sheng Z.H. Neuron. 2000; 25: 191-201Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), α/β-SNAP (9Sollner T. Bennett M.K. 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Science. 1992; 257: 255-259Crossref PubMed Scopus (1085) Google Scholar, 29Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) as well as a growing assortment of channels/receptors (see, for example Refs. 2Inoue A. Obata K. Akagawa K. J. Biol. Chem. 1992; 267: 10613-10619Abstract Full Text PDF PubMed Google Scholar, 3Yoshida A. Oho C. Omori A. Kuwahara R. Ito T. Takahashi M. J. Biol. Chem. 1992; 267: 24925-24928Abstract Full Text PDF PubMed Google Scholar, 30Bezprozvanny I. Scheller R.H. Tsien R.W. Nature. 1995; 378: 623-626Crossref PubMed Scopus (383) Google Scholar, 31Naren A.P. Quick M.W. Collawn J.F. Nelson D.J. Kirk K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10972-10977Crossref PubMed Scopus (129) Google Scholar, 32Leyman B. Geelen D. Quintero F.J. Blatt M.R. Science. 1999; 283: 537-540Crossref PubMed Scopus (188) Google Scholar). Biochemical studies of syntaxin, including structural determinations (8Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar, 33Fiebig K.M. Rice L.M. Pollock E. Brunger A.T. Nat. Struct. Biol. 1999; 6: 117-123Crossref PubMed Scopus (239) Google Scholar, 34Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (625) Google Scholar), have made almost exclusive use of the cytoplasmic domain of the protein. Yet, a number of reports indicate that the TMD of syntaxin is a critical determinant for protein-protein interactions; removal of the TMD inhibits synaptotagmin, synaptobrevin, and α/β-SNAP (25Hanson P.I. Otto H. Barton N. Jahn R. J. Biol. Chem. 1995; 270: 16955-16961Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 29Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) binding activity. In addition, insertion of the transmembrane region into membranes is required for cleavage of syntaxin by botulinum neurotoxin C1 (35Blasi J. Chapman E.R. Yamasaki S. Binz T. Niemann H. Jahn R. EMBO J. 1993; 12: 4821-4828Crossref PubMed Scopus (487) Google Scholar, 36Schiavo G. Shone C.C. Bennett M.K. Scheller R.H. Montecucco C. J. Biol. Chem. 1995; 270: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), and the membrane anchors of syntaxin and synaptotobrevin are required for maximal stability of the SNARE complex (37Poirier M.A. Hao J.C. Malkus P.N. Chan C. Moore M.F. King D.S. Bennett M.K. J. Biol. Chem. 1998; 273: 11370-11377Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To better understand syntaxin protein-protein interactions, we have carried out a detailed investigation of the role of the syntaxin TMD in mediating syntaxin-target protein interactions. We provide evidence that the TMD of syntaxin affects the ability of its cytoplasmic domain to engage partner proteins. The TMD fulfills this role in a length- and sequence-specific manner that is not dependent upon TMD-mediated dimerization. Furthermore, mutagenesis experiments demonstrate that membrane insertion and wild-type partner protein binding activity can be completely uncoupled. We propose that the TMD affects target protein interactions by affecting the conformation of the cytoplasmic domain of syntaxin. Mutagenesis (truncation, deletion, and chimeric protein construction), expression, and purification of recombinant proteins were carried out as described (38Chapman E.R. Jahn R. J. Biol. Chem. 1994; 269: 5735-5741Abstract Full Text PDF PubMed Google Scholar, 39Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). cDNA to generate syx-mult and syx-A15 (19Laage R. Rohde J. Brosig B. Langosch D. J. Biol. Chem. 2000; 275: 17481-17487Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) were kindly provided by D. Langosch (Heidelberg, Germany). cDNA encoding rbSec1A (21Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (226) Google Scholar, 40Garcia E.P. McPherson P.S. Chilcote T.J. Takei K. De Camilli P. J. Cell Biol. 1995; 129: 105-120Crossref PubMed Scopus (243) Google Scholar), syntaxin 1A (4Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1085) Google Scholar), synaptobrevin 2/VAMP2 (41Elferink L.A. Trimble W.S. Scheller R.H. J. Biol. Chem. 1989; 264: 11061-11064Abstract Full Text PDF PubMed Google Scholar), and synaptotagmin I (42Perin M.S. Fried V.A. Mignery G.A. Jahn R. Sudhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (685) Google Scholar) were kindly provided by P. De Camilli (New Haven, CT), R. Scheller (Stanford, CA) and T. C. Sudhof (Dallas, TX). Crude synaptosomes were prepared by homogenization of 1–2 fresh rat brains in 320 mm sucrose buffer. The homogenate was centrifuged at 5000 rpm for 2 min in a Beckman JA-17 rotor; the pellet was discarded, and the crude synaptosome fraction was collected by centrifuging the supernatant at 11,000 rpm for 12 min in the same rotor. The resulting pellet was resuspended in 25–30 ml of 50 mm HEPES, pH 7.4, 100 NaCl buffer plus 1% Triton X-100 and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 20 μg/ml aprotinin) and solubilized for 30–45 min at 4 °C on a rotator. Insoluble material was removed by centrifugation at 17,000 rpm for 20 min in a Beckman JA-17 rotor. The final detergent extract yielded 1 mg/ml protein, and 1-mg aliquots were incubated with 30 μg of immobilized fusion protein as described below. All binding assays were carried out by immobilizing one protein on glutathione-Sepharose beads. Immobilized fusion proteins were incubated with either purified soluble binding partners in Tris-buffered saline (TBS; 20 mm Tris, 150 mm NaCl) plus 0.5% Triton X-100 or rat brain detergent extracts (1 ml at 1 mg/ml, described above) with either 2 mm EGTA or 1 mm free Ca2+ for 1–2 h at 4 °C. Beads were washed three times in binding buffer. Bound proteins were solubilized by boiling in SDS-sample buffer, subjected to SDS-PAGE, and visualized by staining with Coomassie Blue or by immunoblotting. For blotting, mouse monoclonal antibodies directed against synaptotagmin I (604.4 and 41.1), α/β-SNAP (77.1), SNAP-25 (71.2), and synaptobrevin II (69.1) were kindly provided by R. Jahn and S. Engers (Gottingen, Germany). Immunoreactive bands were visualized using enhanced chemiluminescence. Each binding assay was carried out in at least three independent trials, and representative experiments are shown in the figures. Wild-type and mutant syntaxin cDNAs cloned into pGEX-2T were used as PCR templates using a 5′ primer containing the T7 promoter plus sequence complementary to the 5′-end of pGEX-2T. The reverse primer was complementary to the 3′-end of pGEX-2T. PCR was carried out using 30 ng of plasmid DNA, 13.3 μm primers, and Pfu polymerase; samples were cycled 25 times (45 s at 95 °C, 45 s at 54 °C, and 2 min at 72 °C). PCR products (0.5 μg) were then used directly in a TnT in vitro transcription/translation system (Promega, Madison, WI) by incubating with 25 μl of reaction mix containing reticulocyte lysate and [35S]methionine according to the manufacturer's instructions and with canine pancreatic microsomes added as indicated. To determine incorporation of syntaxin into microsomal membranes, 5 μl of the translation mix was added to 400 μl of K-Glu buffer (120 mm potassium glutamate, 20 mm potassium acetate, 2 mm EGTA, 20 mm HEPES, pH 7.2) and, to pellet the membranes, centrifuged at 70,000 rpm for 30 min in a Beckman TLA 100.3 rotor. As indicated, parallel samples were washed with 400 μl of 100 mm Na2CO3buffer, pH 11.5, and membranes were collected by centrifugation as described above. Pellet and supernatant samples were solubilized by boiling in reducing SDS-sample buffer, and equal fractions were subjected to SDS-PAGE. Gels were processed for fluorography using Amplify (Amersham Pharmacia Biotech) and fluorographs are shown in Fig.7. Previous studies indicated that removal of the transmembrane domain (TMD) of syntaxin impaired synaptotagmin, synaptobrevin, and α/β-SNAP binding activity (25Hanson P.I. Otto H. Barton N. Jahn R. J. Biol. Chem. 1995; 270: 16955-16961Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 29Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Furthermore, syntaxin must be anchored into lipid bilayers via its C-terminal membrane-spanning domain to be cleaved by botulinum neurotoxin C (35Blasi J. Chapman E.R. Yamasaki S. Binz T. Niemann H. Jahn R. EMBO J. 1993; 12: 4821-4828Crossref PubMed Scopus (487) Google Scholar, 36Schiavo G. Shone C.C. Bennett M.K. Scheller R.H. Montecucco C. J. Biol. Chem. 1995; 270: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Finally, removal of the syntaxin TMD decreases the stability of fully assembled SNARE complexes (37Poirier M.A. Hao J.C. Malkus P.N. Chan C. Moore M.F. King D.S. Bennett M.K. J. Biol. Chem. 1998; 273: 11370-11377Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Whereas these reports suggested that the TMD of syntaxin is important for protein-protein interactions, it was not clear whether complete removal of the transmembrane segment of syntaxin grossly affected the structure of the protein, or whether the TMD played a more specific or direct role in mediating protein-protein interactions. Therefore, we began to investigate the role of the transmembrane domain in protein-protein interactions by constructing more subtle truncations at the C terminus of the TMD. These constructs, shown in Fig. 1 A, were expressed as GST fusion proteins and used as affinity matrices for the binding of synaptotagmin I, α/β-SNAP, SNAP-25, and synaptobrevin II present in Triton X-100 extracts of rat brain membranes. As shown in Fig. 1 B, truncation of the TMD resulted in the progressive loss of synaptotagmin, α/β-SNAP, and synaptobrevin binding activity. Even removal of the last two amino acids of the TMD slightly, yet reproducibly, reduced synaptotagmin interactions. Truncation to amino acid 281, which would be predicted to lie within the opposite leaflet of the lipid bilayer relative to the cytoplasmic domain, dramatically reduced binding. These data suggest that the distal region of the TMD can affect the interaction of the H3 domain of syntaxin with target proteins. We note that in these experiments, and in experiments described below, similar results were observed using purified recombinant proteins as the "ligands" in the GST pull-down assays (data not shown). Thus, the interactions reported here are direct. In contrast to the diminished binding of synaptotagmin, α/β-SNAP, and synaptobrevin, we observed that SNAP-25 binding was enhanced by removal of the TMD, and this effect became apparent by truncating from amino acid 276 back to residue 271 (Fig. 1 B). These data suggest that truncation of the TMD does not result in gross misfolding, but rather can differentially affect the affinity of different interacting proteins; this effect is consistent with a model in which the TMD truncations can switch syntaxin between different conformations, discussed further below. We also compared the interactions of full-length and the cytoplasmic domain of syntaxin with native (Fig. 1 C) as well as recombinant rbSec1A/nsec-1/munc18 (Fig. 1 D). Analogous to SNAP-25, rbSec1A bound more efficiently to the cytoplasmic domain of syntaxin than to the full-length protein. Thus, removal of the TMD inhibits synaptotagmin, α/β-SNAP, and synaptobrevin binding and enhances SNAP-25 and rbSec1A binding. To determine whether the effects of the syntaxin TMD truncations were length- or position-sensitive, we shortened the TMD by internal deletions at the N-terminal end of the TMD (shown schematically in Fig.2 A). As shown in Fig.2 B, removal of only two amino acid residues at the N-terminal edge of the TMD reduced binding of synaptotagmin and α/β-SNAP. Binding of synaptotagmin was further reduced by progressively larger deletions of four and seven amino acids. Interestingly, synaptobrevin binding was largely unaffected by N-terminal TMD deletions, indicating that these deletions did not result in gross misfolding of syntaxin. Furthermore, SNAP-25 and rbSec1A again showed an increase in binding, and this increase required the removal of seven residues from the N-terminal side of the TMD. For comparison, increased SNAP-25 binding was not observed until more than twelve residues were removed from the C-terminal end of the syntaxin TMD. These data suggest that the role of the TMD in syntaxin binding interactions is complex and involves determinants other than simple length requirements. We addressed this hypothesis via rescue experiments in which we tried to restore wild-type binding interactions with the 1–281 truncation and Δ265–270 deletion mutants by adding the appropriate number of amino acids onto the C-terminal tail of the TMD. To test whether the TMD must form an α-helix of a certain length, we added either a string of isoleucines, which can form an α-helix, or a string of alternating proline/phenylalanine residues (Pro/Phe), which cannot form an α-helix (shown schematically in Fig.3 A). As shown in Fig.3 B, the 1–281 mutant showed diminished synaptotagmin, α/β-SNAP, and synaptobrevin binding activity. Again, SNAP-25 binding was not impaired, providing a positive control for the folding of the mutants. Interestingly, addition of seven Ile residues to the end of the 281 mutant partially rescued α/β-SNAP and synaptobrevin binding, whereas the Pro/Phe sequence did not rescue binding of these proteins. These data indicate that α/β-SNAP and synaptobrevin binding require a full-length TMD with the ability to form an α-helix. In contrast, neither the Ile nor the Pro/Phe sequences rescued synaptotagmin binding, again indicating that the TMD fulfills different requirements for the binding of different interacting proteins. Similar experiments were conducted using the Δ265–270 deletion mutant. Consistent with the data in Fig. 2, this deletion did not affect synaptobrevin binding, but did affect the binding of all other proteins examined (Fig. 3 C). In this case, the Ile sequence failed to rescue synaptotagmin or α/β-SNAP binding whereas, surprisingly, very low levels of rescue were observed with the non-helix forming Pro/Phe sequence. For these interactions, the ability of added on residues to restore wild-type binding activity depended upon whether the TMD was truncated at the N- or C-terminal end, as well as on the content of the added-on sequence. In contrast, the enhanced binding of SNAP-25 to the deletion mutant was partially abrogated by both added-on sequence stretches. In summary, the data from these rescue experiments suggest that the length, primary sequence, and the position of the primary sequence of the TMD of syntaxin are all key factors in syntaxin-target protein interactions. We further examined the sequence requirements of the syntaxin TMD by constructing two chimeric syntaxins that harbored TMDs from synaptobrevin II or synaptotagmin I (Fig.4 A). As shown in Fig.4 B, replacement of the syntaxin TMD with the synaptobrevin TMD resulted in a protein with wild-type, or stronger, synaptotagmin, α/β-SNAP, and synaptobrevin binding activity. In contrast, replacement with the synaptotagmin TMD resulted in only partial rescue of synaptotagmin, α/β-SNAP, and synaptobrevin binding activity. Because the TMDs of synaptobrevin and synaptotagmin are not homologous, these experiments demonstrate some degree of promiscuity in the sequence requirements within the TMD. These observations, coupled to the findings that mutations on the distal side of the membrane anchor (i.e. the N-terminal truncation mutants) can affect protein-protein interactions (Fig. 1 B), prompted us to investigate the possibility that the role of the TMD is to induce oligomerization of the H3 domain of syntaxin to drive normal interactions with other proteins. Indeed, recent studies have demonstrated that the transmembrane domain of syntaxin mediates syntaxin homodimerization as well as binding to the TMD of synaptobrevin (18Margittai M. Otto H. Jahn R. FEBS Lett. 1999; 446: 40-44Crossref PubMed Scopus (78) Google Scholar, 19Laage R. Rohde J. Brosig B. Langosch D. J. Biol. Chem. 2000; 275: 17481-17487Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The same may hold true of the TMD of synaptotagmin, which has been recently shown to contain a novel clustering site within the N-terminal half of the protein (43Bai J. Earles C.A. Lewis J.L. Chapman E.R. J. Biol. Chem. 2000; 275: 25427-25435Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 44von Poser C. Zhang J.Z. Mineo C. Ding W. Ying Y. Sudhof T.C. Anderson R.G. J. Biol. Chem. 2000; 275: 30916-30924Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To determine whether this oligomerization activity lies within the TMD of synaptotagmin, we mapped the N-terminal clustering site. The constructs used for this analysis (shown schematically in Fig.5 A) were immobilized as GST fusion proteins and were assayed for their abilities to bind a His6-tagged fragment of synaptotagmin (residues 1–265). As shown in Fig. 5 B, the soluble synaptotagmin fragment bound to its immobilized counterpart in a Ca2+-independent manner. Removal of the luminal domain did not inhibit binding, however further truncation that removed the TMD strongly reduced binding activity. These data indicate that the TMD of synaptotagmin could directly mediate oligomerization of the protein, but it is also possible that the TMD enables another region of synaptotagmin to homo-oligomerize. We confirmed these results using native synaptotagmin from brain detergent extracts as the ligand (Fig. 5 C). However, in these experiments, Ca2+ facilitated binding, presumably because of Ca2+-triggered oligomerization of the C2B domain of the protein (Refs. 39Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 45Damer C.K. Creutz C.E. J. Neurochem. 1996; 67: 1661-1668Crossref PubMed Scopus (51) Google Scholar, 46Sugita S. Hata Y. Sudhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 47Chapman E.R. Desai R.C. Davis A.F. Tornehl C.K. J. Biol. Chem. 1998; 273: 32966-32972Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 48Desai R.C. Vyas B. Earles C.A. Littleton J.T. Kowalchyck J.A. Martin T.F. Chapman E.R. J. Cell Biol. 2000; 150: 1125-1136Crossref PubMed Scopus (103) Google Scholar).Figure 5The TMD of synaptotagmin I can oligomerize. A, schematic diagram of synaptotagmin (syt) constructs used for this analysis. B andC, the transmembrane domain of synaptotagmin I mediates oligomerization activity. In B, a recombinant fragment of synaptotagmin comprised of residues 1–265, His6-syt-(1–265) (43Bai J. Earles C.A. Lewis J.L. Chapman E.R. J. Biol. Chem. 2000; 275: 25427-25435Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), was incubated (0.5 μm) with the indicated immobilized GST-synaptotagmin fusion proteins for 2 h at 4 °C. Samples were washed three times in binding buffer and analyzed by SDS-PAGE and immunoblotting. Addition of Ca2+ or EGTA did not affect binding activity (data not shown). In C, these experiments were repeated using rat brain detergent extracts as a source of soluble synaptotagmin. Binding assays were carried out as described in the legend to Fig. 1, and bound native synaptotagmin was visualized by immunoblotting and enhanced chemiluminescence. Protein was visualized using an anti-C2B domain antibody that partially cross-reacts with C2A; both native synaptotagmin as well as the immobilized fusion protein are detected. Open arrows indicate the synaptotagmin fusion protein, the closed arrow indicates native synaptotagmin. The increase in binding observed with Ca2+ likely reflects the Ca2+-triggered oligomerization of the C2B domain of the native protein (39Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 45Damer C.K. Creutz C.E. J. Neurochem. 1996; 67: 1661-1668Crossref PubMed Scopus (51) Google Scholar, 46Sugita S. Hata Y. Sudhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133)
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