The transmembrane domain of syntaxin 1A is critical for cytoplasmic domain protein-protein interactions.
Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin protein-protein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, alpha/beta-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a length-dependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins.
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
J L
JL
Lewis
M
M
Dong
C A
CA
Earles
E R
ER
Chapman
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1
D000954:Antigens, Surface; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1