Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans.
SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) 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 results 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.
Johnson
James R
JR
The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown St, Liverpool L69 3BX, UK.
Ferdek
Pawel
P
Lian
Lu-Yun
LY
Barclay
Jeff W
JW
Burgoyne
Robert D
RD
Morgan
Alan
A
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D009435:Synaptic Transmission; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D010750:Phosphoproteins; D050765:Qa-SNARE Proteins; C082636:Unc-18 protein, C elegans; D033921:Vesicular Transport Proteins