The transmembrane domain of syntaxin 1A is critical for cytoplasmic domainprotein-protein interactions

Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the v esicle protein synaptobrevin is a critical step in neuronal exocytosis. Syn taxin is anchored to the inner face of presynaptic plasma membrane via a si ngle C-terminal membrane-spanning domain. Here we report that this transmem brane domain plays a critical role in a wide range of syntaxin protein-prot ein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, alpha/beta -SNAP, and synaptobrevin binding. In contr ast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/n sec-1/munc18 binding. Normal partner protein binding activity of the isolat ed cytoplasmic domain could be "rescued" by fusion to the transmembrane seg ments of synaptobrevin and to a lesser extent, synaptotagmin. However, effi cient rescue was not achieved by replacing deleted transmembrane segments w ith corresponding lengths of other hydrophobic amino acids. Mutations repor ted to diminish the dimerization of the transmembrane domain of syntaxin di d not impair the interaction of full-length syntaxin with other proteins. F inally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembran e domain, via a length-dependent and sequence-specific mechanism, affects t he ability of the cytoplasmic domain to engage other proteins.