Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

James R Johnson , Pawel Ferdek , Lu-Yun Lian , Jeff W Barclay , Robert D Burgoyne , Alan Morgan (1)

1 University of Liverpool

SNAP receptors (SNAREs) are widely accepted to drive all intracellular membrane fusion events. Sec1/Munc18 (SM) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least 3 modes: to a closed conformation of syntaxin; to the syntaxin N-terminus; and to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore, high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18 null mutant worms were transformed with wild type or mutant unc-18 constructs. The UNC-18(R39C) construct that is defective in closed syntaxin binding fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct that is defective in binding to the N-terminus of UNC-64 provided no rescue. These data suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

Mots clés: Life Sciences

James R Johnson, Pawel Ferdek, Lu-Yun Lian, Jeff W Barclay, Robert D Burgoyne, et al.. Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans. Biochemical Journal, 2009, 418 (1), pp.73-80. ⟨10.1042/BJ20081956⟩. ⟨hal-00479101⟩