Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Aug 15;97(17):9695-700.
doi: 10.1073/pnas.97.17.9695.

Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

S Quetglas  1 , C Leveque, R Miquelis, K Sato, M Seagar
Affiliations

Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

S Quetglas et al. Proc Natl Acad Sci U S A. .

Abstract

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca(2+) could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca(2+)-dependent calmodulin binding (K(d) = 500 nM) to glutathione S-transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP(77-94), immediately C-terminal to the tetanus toxin cleavage site (Q(76)-F(77)). In isolated synaptic vesicles, Ca(2+)/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a (35)S-SNAP-25, syntaxin 1 GST-VAMP(1-96) complex was inhibited by Ca(2+)/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca(2+) may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Calcium-dependent calmodulin binding to GST-VAMP 21–96. (A) Bacterially expressed GST-fusion proteins containing SNAP-25, the cytoplasmic domains of syntaxin 1A, VAMP 2, and GST (2 μM) were incubated with calmodulin–agarose (5 μM) in TBS in the presence of 1 mM CaCl2 or 5 mM EDTA. After extensive washing, bound proteins were eluted with sample buffer and analyzed by SDS/PAGE and Coomassie blue staining. (B) GST-VAMP 21–96 was diluted into running buffer containing 1 mM CaCl2 and injected on a streptavidin-coated sensorchip loaded with biotinylated calmodulin. An increase in resonance occurred during VAMP 2 injection (association), followed by decrease at the end of the injection period (dissociation). Injection of EDTA induced an abrupt return to baseline (regeneration). Experiments with a range of VAMP 2 concentrations allowed calculation of the rate constants and Kd reported in the text.
Figure 2
Figure 2
Localization of the calmodulin-binding site to the C-terminal domain of VAMP 2. (A) Residues 77–90 of rat VAMP 2 contain a 1–5-8–14 consensus motif for calcium-dependent calmodulin binding proposed by Rhoads and Friedberg (18). A comparison of VAMP homologues from rat (VAMP 3/cellubrevin), sea urchin (Strongylocentrotus purpuratus) cortical vesicles, aplysia (Aplysia californica), nematode (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae Snc1) illustrates evolutionary conservation of this motif. Basic residues within the motif are underlined. Two conserved lysines (+) C-terminal to the motif also contribute to calmodulin interactions (see Results). (B) The binding of GST-fusion proteins containing the sequences VAMP1–96, VAMP1–96 cleaved with TeTx light chain (i.e., VAMP1–76), VAMP77–90, and VAMP1–90 to calmodulin–agarose beads was assayed as in Fig. 1A. (C) Immunoisolated synaptic vesicles from rat brain were incubated with TeTx light chain (300 pM) in buffer containing 1 mM CaCl2, in the presence or absence of calmodulin (15 μM). Duplicate aliquots were removed immediately (0 min) or after a 30-min incubation. Samples were denatured and processed for Western blotting with an antibody directed against the N-terminal region of VAMP 2.
Figure 3
Figure 3
Inhibition of SNARE complex assembly by calcium/calmodulin. (A) In vitro translated 35S-SNAP-25 was incubated in the presence or absence of GST, GST-VAMP1–96, and untagged syntaxin 1A1–261 for 3 h at 4°C. Protein complexes were recovered on glutathione beads and, after washing, bound 35S-SNAP-25 was measured by β counting. Results are means ± SD, n = 3. (B) Samples prepared as in A were analyzed by SDS/PAGE and autoradiography after denaturation in SDS at 37°C or 100°C. Arrows indicate the migration of the trimeric core complex (upper arrow) and 35S-SNAP-25 (lower arrow). The radioactive band at about 40 kDa in the first two lanes is an unidentified translation product that did not interact with VAMP 2 or syntaxin 1A. (C) 35S-SNAP-25, GST-VAMP1–96, and syntaxin 1A1–261 were incubated at 4°C in a buffer containing 1 mM CaCl2, in the presence or absence of calmodulin (10 μM). At the indicated times, samples were removed, and bound 35S-SNAP-25 was evaluated by β counting. The curve is representative of three independent experiments. (D) SNARE assembly was performed as in A, except that complexes were recovered on calmodulin–agarose beads in the presence or absence of 1 mM CaCl2 and after pretreatment of GST-VAMP1–96 with TeTx light chain. 35S-SNAP-25 retained by calmodulin–agarose is shown as a percentage, taking the radioactivity recovered on glutathione beads as 100%, means + SD, n = 3.
Figure 4
Figure 4
Interactions of phospholipids with the calmodulin-binding domain of VAMP 2. (A) GST-fusion proteins containing the indicated VAMP sequences were immobilized on an CM5 (Biacore) sensor chip coated with anti-GST antibodies. GST-VAMP1–76 was prepared from VAMP1–96 by TeTx cleavage. Liposomes containing 7.5 dipalmitoyl-PC: 2.5 dipalmitoyl-PS; weight: weight (PS+), or pure dipalmitoyl-PC (PS−), were diluted in running buffer and injected in the presence or absence of a synthetic peptide corresponding to VAMP 2 residues 77–94 (p77–94). Experiments were performed in the presence of 1 mM CaCl2 or 5 mM EDTA. Results are shown as means ± SD, n = 3. (B) Biotinylated calmodulin was immobilized on a streptavidin-coated sensorchip, and GST-VAMP1–96 was injected in the absence or presence of PC/PS liposomes. Control injections of liposomes were carried out in the absence of VAMP.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sollner T, Bennett M K, Whiteheart S W, Scheller R H, Rothman J E. Cell. 1993;75:409–418. - PubMed
    1. Sollner T, Whiteheart S W, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman J E. Nature (London) 1993;362:318–324. - PubMed
    1. Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta B R, Montecucco C. Nature (London) 1992;359:832–835. - PubMed
    1. Niemann H, Blasi J, Jahn R. Trends Cell Biol. 1994;4:179–185. - PubMed
    1. Sutton R B, Fasshauer D, Jahn R, Brunger A T. Nature (London) 1998;395:347–353. - PubMed

MeSH terms