A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Abstract

The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicleassociated SNARE synaptobrevin 2 that lie close to the cytosol-membrane interface within the full-length protein.Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion.

Transmembrane-domain determinants for SNARE-mediated membrane fusion

“…For this purpose, neuroendocrine PC12 cells were transfected with a plasmid containing both non-tagged VAMP2 and human growth hormone (hGH) (Fdez et al, 2008). Cells were permeabilised and treated with recombinant botulinum neurotoxin type F light-chain (botF/LC), which cleaves and inactivates VAMP2 (Fig.…”

“…For secretion assays, full-length, non-tagged rat VAMP2 (residues 1-116) was expressed from a pCMV-driven promoter in a vector where expression of hGH is driven from an SV40 promoter (pCMV5-VAMP2-SV40-hGH) (Fdez et al, 2008). C-terminally GFP-tagged VAMP2 was generated by subcloning full-length VAMP2 into a pGFPemd vector (Packard) using the EcoRI-BamHI sites, with a resulting linker (TDPPVAT) between the VAMP2 and the GFP sequences.…”

“…PC12 cells were grown and transfected essentially as described (Fdez et al, 2008). Double transfections were performed with each 2.4 g DNA using 10 l LipofectAMINE 2000 (Invitrogen).…”

“…Cells were examined either live, or upon fixation (Fdez et al, 2008) and mounting (ProLong-Gold antifade reagent, Invitrogen). For colocalisation analysis, the following antibodies were used: rabbit polyclonal antisynaptotagmin (p65, 1:1000) (Fdez et al, 2008), mouse monoclonal anti-VAMP2 (Cl 69.1, Synaptic Systems, 1:5000), mouse monoclonal anti-rat TGN38 (BD Biosciences, 1:800), rabbit polyclonal anti-calnexin (Stressgen, SPA860, 1:300), mouse monoclonal anti-syntaxin-(CL HPC-1, SIGMA, 1:500), mouse monoclonal anti-TfR (Invitrogen, 1:300), mouse monoclonal anti-EEA1 (BD Biosciences, 1:250), mouse monoclonal anti-synaptophysin (CL SVP38, Sigma, 1:200), mouse monoclonal anti-GM130 (BD Biosciences, 1:100) and rabbit polyclonal anti-GFP (Abcam, 1:500). Secondary antibodies included Alexa Fluor 594-conjugated goat anti-mouse (Invitrogen, 1:1000) and Alexa Fluor 594-conjugated goat anti-rabbit (Invitrogen, 1:1000).…”

“…Distinct molecular interactions responsible for oligomerisation of SNARE complexes have been reported (Littleton et al, 2001;Tokumaru et al, 2001;Kweon et al, 2002;Rickman et al, 2005;Fdez et al, 2008), including interactions mediated by the transmembrane domains (TMDs). For example, self-interaction of the syntaxin-1A TMDs has been proposed to have a scaffolding role for the subsequent formation of a supramolecular SNARE complex at the fusion site (Lu et al, 2008), or to be implicated in mediating the transition from a hemifusion to a full-fusion state (Hofmann et al, 2006).…”