Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion.
Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.
INSERM/Université de la Méditerranée UMR 464, IFR Jean Roche, Faculté de Médecine secteur Nord, Bd Pierre Dramard, 13916 Marseille cedex 20, France.
Luc
L
De Haro
Stéphanie
S
Quetglas
Cécile
C
Iborra
Christian
C
Lévêque
Michael
M
Seagar
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism / Q000502:physiology*; D020134:Catalytic Domain; D019439:Chromaffin Cells / Q000201:enzymology; D016384:Consensus Sequence; D005089:Exocytosis; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*
D002147:Calmodulin; D008565:Membrane Proteins; D010743:Phospholipids; D050683:R-SNARE Proteins