SNAP-25 Functional Domains in SNARE Core Complex Assembly and Glutamate Release of Cerebellar Granule Cells

Abstract

Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181-206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1-31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30 -35% inhibition is obtained with the Cterminal deletion mutant, and the inhibitory effect is abolished in the N-and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons. SNAP-251 is a major component of the so-called SNARE complex that is involved in vesicle docking and fusion at synaptic junctions (1-3). The core complex includes the SNARE proteins syntaxin, SNAP-25, and synaptobrevin (vesicle-associated membrane protein). It has been shown by in vitro binding studies that the protein interacts directly with syntaxin, synaptobrevin, and synaptotagmin (4 -8). SNAP-25 is localized primarily to plasma membranes (9, 10), and it interacts with membranes through posttranslational palmitoylation (11). The membrane binding domain of SNAP-25 contains four cysteine residues and mutation of two or more cysteines effectively abolishes membrane binding (12). The syntaxin binding domain is located in the N terminus of .Recently the crystal structure of the SNARE core complex was solved (13). It shows that the N-and C-terminal helix domains of SNAP-25 participate in the formation of a parallel coiled-coil structure with syntaxin and synaptobrevin (13-16). By limited proteolysis and in vitro binding assay, it is proposed that the two ␣-helix domains act independently and contribute equally to form the SNARE complex with syntaxin and synaptobrevin (14). These results provide further understanding into how SNAP-25 may participate in the assembly of SNARE complex and vesicle fusion.Although the importance of SNAP-25 and the SNARE complex in neurosecretion is well documented, the molecular mechanism underlying their involvement in the membrane fusion process is less clear. Recent studies of yeast vacuole fusion and the cortical vesicle fusion in sea urchin eggs showed that membrane fusion can proceed in the absence of SNARE formation (17, 18), indicating that the SNARE complex may not directly participate in, and thus may not be required for actual membrane fusion. On the other hand, the formation of the SNARE complex ...

Localization and Function of SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein-25 and Vesicle-associated Membrane Protein-2 in Functioning Gastric Parietal Cells

“…In addition, our binding data demonstrate that the C-terminal deletion mutant unlike wild type SNAP-25 was relatively ineffective in associating with syntaxin-1. These secretion and binding data agree with a previous study involving cerebellar neurons, which showed that overexpressed SNAP-25 ⌬180 -206 mutant altered the vesicle fusion kinetics, reaffirming the importance of SNAP-25 and specifically this C-terminal segment in exocytosis (12). The results also add further evidence for the importance of SNARE proteins for the activation of parietal cells.…”

“…These results suggest that SNAP-25 may promote membrane fusion by contributing two cytoplasmic-helices including the C-terminal helix to stabilize the four-helix core complex. Yang et al (12) recently report that the overexpression of the C-terminal deletion mutant ⌬180 -206 significantly inhibited glutamate release from neuronal cells. Because of the functional effects in neuronal cells, we used the C-terminal deletion mutant to study the function of SNAP-25 in parietal cells.…”

“…In Vitro SNARE Core Complex Formation-Recombinant rat GSTsyntaxin-1 was expressed in bacteria as fusion protein as described previously (12). Wild type and mutant SNAP-25 were expressed in gastric glands for 18 h by rAd infection, which were then solubilized with Triton X-100 and used for binding assay.…”

“…Cells were then scraped from the coverslip, and the protein content of the cell scrapings was assayed using the filter paper blot method (10). Aliquots of both the reaction medium and cell scrapings were assayed for [ 14 Adenoviral Infection of Gastric Parietal Cells and Gastric GlandsAdenoviruses were obtained as previously described with the following mouse cDNA constructs (11,12): SNAP-25 fused to cyan fluorescent protein (CFP); VAMP-2 fused to yellow fluorescent protein (YFP); SNAP-25 wild type and GFP unfused; and C-terminal deletion mutant SNAP-25 ⌬181-206 and GFP unfused. Adenoviruses with the incorporated cDNAs (rAd) were used to infect cells and glands with the respective cDNAs.…”

Identification of Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for Antibody Secretion

“…The inhibitory effect of GST-SNAP-23del1-203 on PC Ig secretion is consistent with the results observed in 3T3-L1 adipocytes infected with recombinant adenoviruses carrying SNAP-23delC8 (48). The explanation of how GST-SNAP-23delC8 inhibits cell secretion remains unknown, but it has been suggested that a competition could exist between the deleted protein and the endogenous SNAP-23, resulting in either the formation of unstable SNARE complexes of GST-SNAP-23delC8 with the other SNARE partners (49,50), or the prevention of endogenous SNAP-23 from forming a SNARE complex (51). The finding that undeleted GST-SNAP-23 also produces an identical inhibition of PC Ig secretion indicates that additional events might explain the present results.…”