A Central Small Amino Acid in the VAMP2 Transmembrane Domain Regulates the Fusion Pore in Exocytosis

Benoît Hastoy,Alexandra Milochau,Jochen Lang,Pier A Scotti,Rémi Megret,Sabine Castano,Reiko Oda,Bernard Desbat,David Perrais,Jorge Rodas,Zahia Fezoua-Boubegtiten,Michel Laguerre,Patrik Rorsman

doi:10.1038/s41598-017-03013-3

Abstract

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with the β-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/β-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that G100V/C103V retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.

Highlights

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial
We combined a structural analysis of VAMP2 transmembrane domain dynamics and a characterization of its potential cellular function
Biochemical, electrophysiological and optical approaches demonstrate that VAMP2 modified in the TMD region (G100V/C103V) is unable to restore exocytosis

Summary

Introduction

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. Substituting G100 and/or C103 with the β-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/β-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. PC12 cells were transiently co-transfected with a plasmid expressing the human growth hormone (hGH), shC or shV, and the indicated VAMP2R (WTR or mutant in the TMD). The latter observations raise the interesting possibility that the TMDs may play a role in exocytosis that extends beyond being merely a membrane anchor This is further supported by a recent work from Dhara and colleagues who replaced half of VAMP2 TMD and showed altered fusion pore kinetics[10]

Methods

Results

Conclusion