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

Colin Rickman,Frederic A Meunier,Thomas Binz,Bazbek Davletov

doi:10.1074/jbc.m310879200

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.

Highlights

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane
We 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
Where A(SNAP-25/syntaxin1) is the area occupied by SNAP-25 clusters that colocalize with syntaxin1, A(SNAP-25) is the area occupied by SNAP-25 clusters, %FC is the percentage of full colocalization between SNAP-25 and syntaxin1, %NC is percentage of neutral colocalization between SNAP-25 and syntaxin1, and A(TotChr) is the average total area of the plasma membrane of chromaffin cells estimated to be 801.6 ␮m2 (n ϭ 120)

Summary

EXPERIMENTAL PROCEDURES

Preparation of Proteins—Plasmids encoding glutathione S-transferase (GST) fusion proteins with syntaxin (amino acids 1–261) and synaptobrevin (amino acids 1–96) were described previously [10]. Recombinant SNARE proteins, purified on glutathione-Sepharose beads (Amersham Biosciences), were washed with buffer A (20 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM EDTA) followed by elution with 15 mM reduced glutathione in buffer A or thrombin cleavage. Beads were washed three times by low speed centrifugation with 1 ml of buffer A, and bound protein was eluted in SDS-containing sample buffer followed by SDSPAGE and Coomassie staining. The beads were washed three times with 1 ml of buffer A before analysis of bound protein by SDS-PAGE and Coomassie staining. For determination of the stoichiometry of SNAREs in the syntaxin1/ SNAP-25 heterodimer, proteins bound to the beads were eluted into sample buffer and separated by SDS-PAGE. Where A(SNAP-25/syntaxin1) is the area occupied by SNAP-25 clusters that colocalize with syntaxin, A(SNAP-25) is the area occupied by SNAP-25 clusters, %FC is the percentage of full colocalization between SNAP-25 and syntaxin1, %NC is percentage of neutral colocalization between SNAP-25 and syntaxin, and A(TotChr) is the average total area of the plasma membrane of chromaffin cells estimated to be 801.6 ␮m2 (n ϭ 120)

RESULTS

Target SNARE Heterodimers on the Plasma Membrane

DISCUSSION