The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusi on machinery. Syntaxin probably interacts with multiple proteins during syn aptic vesicle exocytosis, In vitro, the tightest binding partners for synta xin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1), Recent studies on Drosophila syntaxin led to the su rprising finding that a syntaxin mutant which does not bind the munc18-homo log Rop nevertheless functionally substitutes for wild-type syntaxin in mem brane fusion (Wu et al,, Neuron 23, 593-605, 1999), This observation sugges ted that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzz ling conclusion in view of the tight binding of munc18 and syntaxin homolog s in all organisms. To address this issue, we have now reinvestigated the i nteraction of syntaxin with munc18 and Rep. We find that the syntaxin seque nce that was mutated in the Drosophila studies is not essential for munc18/ Rop binding, and that the mutant is in fact able to bind to munc18/Rop, Thu s the fact that the mutant syntaxin rescues release cannot be used as an ar gument that munc18 binding is not essential. In addition to munc18 and SNAR Es, several other proteins have been suggested to interact with various dom ains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicl e protein CSP Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus o f the motif. Interestingly, binding of synaptotagmin appears to be decrease d in the closed conformation of syntaxin, In contrast, no interaction of CS P with syntaxin was detected even under low-stringency conditions. Our data suggest 1,, that assays measuring protein/protein interactions that involv e syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2,, that it is currently not possible to draw conclusions about the importance of the various inter actions with the available data from Drosophila or vertebrates.