The Transmembrane Domain of Syntaxin 1A Is Critical for Cytoplasmic Domain Protein-Protein Interactions

Abstract

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 proteinprotein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, ␣/␤-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 lengthdependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins.Syntaxin 1A was initially identified as a 35 kDa protein in the plasma membrane of amacrine cells (1), as a subunit of Ca 2ϩ channels (2, 3) and as a synaptotagmin-binding protein (4). Since these initial reports, the function of syntaxin as a central component in the synaptic vesicle membrane fusion machinery has been well established (reviewed in Refs. 5-7). Syntaxin forms a putative membrane fusion apparatus by assembling into a fourhelix bundle (8) with the plasma membrane protein SNAP-25 1 and the synaptic vesicle protein synaptobrevin, to form a SNARE complex (9). Assembly of this complex is necessary (10, 11) and may be sufficient to drive membrane fusion (12)(13)(14). One current view is that the zippering together of the four-helix bundle drives membrane fusion by pulling the vesicle and target membranes together (8,12,15). In this model, the transmembrane domains (TMDs) of synaptobrevin and syntaxin would form part of a fusion pore (16). Thus, structure-function relationships of these TMDs may reveal insights into the mechanism of membrane fusion (13,14,(17)(18)(19).Syntaxin functions as a key element in membrane traffic and membrane fusion by interacting with a wide range of other proteins. The many binding partners of syntaxin, in excess of twenty, include rbSec1A/nsec-1/munc18 (20 -22), CSP (23), syntaphilin (24), ␣/␤-SNAP (9, 25), sec6/8 (26), tomosyn (27), Munc-13 (28), and as mentioned above synaptotagmin (4, 29) as well as a growing assortment of channels/receptors (see, for example Refs. 2, 3, 30 -32).Biochemical studies of syntaxin, including structural determinations (8, 33, 34), have made almost exclusive use of the ...

Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport

“…However, because of its mis-localization (data not shown), the H3 domain by itself could not induce EAAC1 inhibition without the assistance of the transmembrane domain (Kasai and Akagawa, 2001;Lewis et al, 2001). In fact, our data show that, at least in the presence of the H3 and transmembrane domains, syntaxin 1A associated with EAAC1 on the cell surface, leading to the reduction of its surface expression and the inhibition of glutamate transport.…”

Binding of Sly1 to Sed5 enhances formation of the yeast early Golgi SNARE complex

“…Recently, it has been reported that the transmembrane domain of syntaxin 1 is critical for its proper interaction with other proteins (Lewis et al, 2001). Actually, Sly1 did not have an effect on the binding after membrane solubilization by Triton X-100 in our cell free system (data not shown).…”

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

“…Pull-down assays GST-pull down assays were performed as described previously using 80 µg bead-immobilized GST-C2ABC or GST-C2DEF (Lewis et al, 2001;Dong et al, 2003). In brief, SNARE complexes were formed by incubating 50 µM t-SNARE heterodimer and 50 µM synaptobrevin 2 overnight at 4°C.…”

“…Gels were digitized using a scanner and processed using Illustrator. Whole brain lysate immunoprecipitation assays were performed using rat brain detergent extracts that were prepared as described previously (Chapman et al, 1995;Lewis et al, 2001). 1% Triton X-100 was used for solubilization.…”