Protease Resistance of Syntaxin·SNAP-25·VAMP Complexes

Abstract

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A⅐SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and aminoand carboxyl-terminal regions of SNAP-25. Furthermore, separate amino-and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino-and carboxyl-terminal regions of SNAP-25 may function as independent domains.Neurotransmitter release represents a specialized form of regulated secretion wherein neurotransmitter-containing synaptic vesicles selectively dock and fuse with the plasma membrane. This process constitutes an essential step in chemical synaptic transmission and is a likely target for regulatory reactions that modulate the strength of synaptic signaling. The identification and biochemical characterization of numerous synaptic vesicle and presynaptic plasma membrane proteins have provided fundamental insight into the molecular mechanisms underlying neurotransmitter release (for reviews see Refs 1 and 2).Among the important findings that have emerged from molecular studies of neurotransmitter release is the central role of a protein complex composed of two presynaptic plasma membrane proteins, syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), 1 along with one synaptic vesicle protein, vesicle-associated membrane protein 2 (VAMP2; also known as synaptobrevin). Several lines of evidence suggest a primary role for syntaxin 1, SNAP-25, and VAMP2 in synaptic vesicle exocytosis. First, each represents a substrate for cleavage by distinct clostridial neurotoxins, toxins that irreversibly inhibit neurotransmitter release in vivo (3, 4). Furthermore, genetic studies in Drosophila have shown that loss of syntaxin 1 or VAMP2 prevents Ca 2ϩ -dependent exocytosis (5). Finally, the ternary complex formed by all three proteins acts as a receptor for soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs), cytosolic proteins required in multiple membrane trafficking events (6). Consequently, the syntaxin 1⅐SNAP-25⅐VAMP2 ternary complex is commonly referred to as the synaptic SNA...

SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

“…3i, amino acids 85-120 of SNAP-25 were sufficient to target a heterologous protein to the plasma membrane of NG108 cells. Interestingly, this sequence coincides almost exactly with the protease-sensitive interhelical domain (residues 83-120) of SNAP-25 in the SNARE complex (11,12). All of the deletion constructs were expressed at similar levels and migrated at the predicted molecular mass as assessed by immunoblotting (data not shown).…”

“…The cysteine-rich sequence is contained within the linker between the N-and C-terminal helices that SNAP-25 contributes to the core synaptic fusion complex (4). The structure of the linker domain is unknown, but is presumed to be exposed and disordered in the complex in vitro because of its susceptibility to proteolytic cleavage (11,12). In any case, this region of SNAP-25 has to be sufficiently extended to accommodate the parallel orientation of both SNAP-25 helices in the synaptic fusion complex (4).…”

SNARE proteins contribute to calcium cooperativity of synaptic transmission

“…Poirier et al (45) have suggested that SNAP-25 may contribute to SNARE complex oligomerization if its C-terminal ␣-helix participates in a different SNARE complex than its N-terminal ␣-helix. Other proteins that interact with the SNARE complex, such as complexin, or Ca 2ϩ -dependent oligomerization of synaptotagmin also may mediate this function.…”

Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

“…Moreover, inclusion of these TMDs increased the stability of the ternary complexes and affected the ability of the cytoplasmic domains to join other proteins (4,5). Co-reconstituted into liposomes, synaptobrevin II and syntaxin 1A formed a stable binary complex that required the presence of the TMDs (6).…”

“…Analysis of ternary complexes formed by the full-length proteins revealed that the C-terminal transmembrane domains (TMDs) of both Sx1A and synaptobrevin II were protected from trypsin digestion (4). Moreover, inclusion of these TMDs increased the stability of the ternary complexes and affected the ability of the cytoplasmic domains to join other proteins (4,5).…”