Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Robert D Lagow,Hong Bao,Bing Zhang,Richard W Daniels,Evan N Cohen,R Bryan Sutton,Gregory T Macleod,Wade H Williams,Aleksej Zuzek

doi:10.1371/journal.pbio.0050072

Abstract

Both constitutive secretion and Ca2+-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Highlights

Soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) proteins are thought to mediate vesicle fusion in all eukaryotes [1,2,3,4]
We found that a mutant SNARE protein, syntaxin at the synapse, contained a building block commonly conserved for syntaxins functioning along constitutive secretory pathways
Our modeling predicted that the mutant syntaxin could form a tightly packed SNARE bundle closely resembling that found in the endosome, but differing from the relatively loosely packed bundle found at the wild-type synapse

Summary

Introduction

Soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) proteins are thought to mediate vesicle fusion in all eukaryotes [1,2,3,4]. There are two target-SNAREs (t-SNAREs, called Q-SNAREs), syntaxin 1A and synaptosome-associated protein-25 kDa (SNAP-25) on the plasma membrane, and one vesicle-associated SNARE (v-SNARE, called R-SNARE), synaptobrevin 2 on synaptic vesicles [2]. The t- and v-SNAREs are thought to form a trans complex composed of a four-stranded helical bundle with one helix each from syntaxin and synaptobrevin and two helices contributed by SNAP-25 [5,6,7,8,9] (Figure 1A). The SNARE complex rearranges from a trans to a cis configuration such that all the SNARE proteins are localized to one membrane. The specific mechanism of vesicle fusion is still in debate, it is widely accepted that the formation of this four-helix bundle is essential for the fusion of the vesicle phospholipid bilayer with the plasma membrane phospholipid bilayer [3]

Methods

Results

Discussion

Conclusion