High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Abstract

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.Hormonal and neurotransmitter release occurs when vesicles fuse with the plasma membrane in a calcium-dependent manner. The three SNARE 1 proteins, namely synaptobrevin2 (also known as VAMP2) on the vesicular membrane and syntaxin1 and SNAP-25 on the target plasma membrane, are essential for the last step of vesicular exocytosis, the fusion of membranes (1, 2). These three proteins form a highly stable ternary complex that likely drives the fusion of the two apposing membranes (3). Botulinum toxins, which specifically cleave SNARE proteins prior to the formation of the ternary SNARE complex, block vesicle exocytosis in both neurons and endocrine cells (2, 4). The plasma membrane protein SNAP-25 is a molecular target for botulinum toxin E (5). This toxin removes the C-terminal 26 amino acids from the SNAP-25 molecule and has been used previously to dissect stages of calcium-triggered exocytosis (6, 7). Although these studies demonstrated that the ternary SNARE complex forms at a late stage of vesicle exocytosis, the molecular events preceding formation of this complex remain largely unknown.Several contradicting theories have been put forward with regard to the molecular pathway leading to SNARE complex assembly. One study suggested that SNAREs, although active, do not pre-assemble in resting cells into any intermediate complex but would form the ternary complex upon activation of exocytosis (8). It has also been proposed that SNAP-25 engages vesicular synaptobrevin in a priming step and that binding of syntaxin1 would be the pivotal event leading to membrane fusion (9). An alternative theory states that syntaxin1 and SNAP-25 pre-assemble into a stable heterodimer on the plasma membrane, and only then does vesicular synaptobrevin engage the two target SNAREs (10, 11). Importantly, it is not clear whether any SNARE intermediates actually exist in intact secretory cells as a prelude to SNARE-mediated fusion.To address these outstanding issues, we analyzed SNARE binary re...

Conserved Prefusion Protein Assembly in Regulated Exocytosis

“…It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al, 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two plasma membrane proteins are together and not apart (Hayashi et al, 1994).…”

“…With all the major players involved in vesicle fusion identified, it is becoming possible to tackle a central problem of this cellular process-how does calcium trigger vesicle fusion? Arguably, to understand the action of calcium it is essential to know 1) the extent of the assembly of the SNARE fusion proteins and 2) their organization in relation to the calcium sensor, SYT, before calcium-triggered events.It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al, 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two plasma membrane proteins are together and not apart (Hayashi et al, 1994).…”

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

“…Studies of the proteins in solution are consistent with this structure (Fasshauer et al, 1997;Lin and Scheller, 1997), which may reflect a configuration of the SNAREs during or after fusion. Physiological interactions of SNAP-25 and syntaxin in the plasma membrane probably occur with a 1:1 ratio (Fasshauer, 2003;Rickman et al, 2004), although in vitro studies of soluble SNARE motifs also demonstrate a 2:1 binding stoichiometry (Fasshauer et al, 1997;Xiao et al, 2001). Little is known about the interactions of membranebound SNAREs in living cells and the relationships of the interactions to secretion.…”

A New Platform to Study the Molecular Mechanisms of Exocytosis

“…It starts with the formation of the binary SNARE complex, between SNAP-25 and syntaxin I, which is the first intermediate protein complex in the model (Lang et al, 2001;Fasshauer and Margittai, 2004;Rickman et al, 2004). The complex later binds synaptobrevin/VAMP II to form the mature ternary SNARE complex (Sutton et al, 1998;Weber et al, 1998;Margittai et al, 2003).…”

“…Similarly, for reaction number i (where i represents the number of the reaction and accordingly can vary from 1 to 15) k on ϭ k i in the differential rate equations. Data are from the following references: (a), Ashery et al, 2000;, Margittai et al, 2003;, Rickman et al, 2004;, Lang et al, 2001;Graham et al, 2004; (e), Lang et al, 2001; (f), Weninger et al, 2003;(g), Pabst et al, 2002;(h), Voets, 2000. forms the SNARE* complex. A novel step in which Munc13-1 is preactivated by Ca 2ϩ was added to the flow chart and accounts for the finding that Munc13 proteins have several calciumbinding domains (Brose et al, 1995;X.…”