SNAP-25 Is Required for a Late Postdocking Step in Ca2+-dependent Exocytosis

Abstract

Neurotransmitter and peptide hormone secretion require fusion between secretory vesicles and the plasma membrane, an exocytotic process activated by cytoplasmic Ca 2ϩ elevation. Understanding regulated secretion requires identification of molecular components that mediate docking and fusion reactions and delineation of rate-limiting steps that are Ca 2ϩ -regulated (1). The synaptic vesicle protein synaptobrevin and the presynaptic membrane proteins syntaxin and SNAP-25 1 are required components of the exocytotic apparatus as indicated by the inhibitory action of BoNTs and Tetx, which involves the specific endoproteolytic cleavage of these protein substrates (2, 3). The three toxin substrates were independently identified as receptors for SNAPs, proteins required for the membrane binding of NSF, a late acting component in constitutive membrane fusion reactions (4). A characterized ternary complex containing synaptobrevin, syntaxin, and SNAP-25 was suggested to represent a docking complex that mediates the targeting, docking, or fusion of secretory vesicles (5, 6). Identification of an ATP-dependent catalytic activity of NSF/SNAP that promotes the disassembly of ternary complexes in vitro led to the suggestion that a similar reaction in vivo was responsible for late steps in membrane fusion (7). Ca 2ϩ -triggered secretion in permeable neuroendocrine cells requires ATP; however, the requirement for ATP precedes that for Ca 2ϩ (8, 9). ATP hydrolysis is required for prefusion events that prime the exocytotic apparatus, whereas Ca 2ϩ -activated fusion proceeds in the absence of ATP (8 -11).2 In priming, ATP serves as a substrate for polyphosphoinositide synthesis (10, 11) and as a substrate for the SNAP-dependent ATPase activity of NSF that catalyzes rearrangement of docking protein complexes.2 These studies experimentally identify a late step in the exocytotic pathway beyond LDCV docking and ATP utilization that is proximal to Ca 2ϩ -dependent fusion reactions. To characterize events that lead to or are directly involved in membrane fusion, it is important to identify molecular components that act at this late Ca were permeabilized by passage through a ball homogenizer (12). Secretion assays were conducted either as single stage or as two-stage assays (9,14). For the former, permeable cells were incubated for 15 min at 30°C in KGlu-BSA buffer supplemented with CaCl 2 (to achieve 10 M Ca 2ϩ f ), 0.002 M MgATP, and 0.5 mg/ml rat brain cytosol. Two-stage assays were conducted as separate priming incubations (30 min at 30°C in KGlu-BSA buffer supplemented with 0.002 M MgATP plus 1 mg/ml rat brain cytosol) followed by triggering incubations (1-3 min at 30°C in KGlu-BSA buffer supplemented with 10 M Ca 2ϩ plus 0.5 mg/ml rat brain cytosol) with extensive washing between incubations. [ 3 H]NE release was determined by centrifugation of permeable cells at 800 ϫ g for 30 min and scintillation counting of 3 H in supernatants and in cell pellets to express NE release as percent of the total 3 H. The SNAP-25 antibody used to inhi...

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

“…4). This observation agrees with an earlier study that showed that botulinum toxin E treatment cleaved the C-terminal 180 -206 sequence of SNAP-25 and destabilized the SNARE complexes in PC12 cells (32). It is also possible, however, that the inhibition was caused by other factors present in the COS-7 cell lysate, even though the cell lysate alone did not seem to affect the SNARE complex assembly/disassembly (data not shown).…”

“…Several elegant studies using synaptosome preparations and permeabilized PC12 cells have suggested that SNAP-25 may act in the late post-docking steps of exocytosis (20,(32)(33)(34). However, there are many questions that can only be answered by directly investigating neurons in vivo, especially at their functional synaptic junctions.…”

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

“…Whether syntaxin is involved in docking has been challenged by the finding that genetic knock-out of Drosophila syntaxin failed to impair vesicle docking . Furthermore, it has been documented that in PC12 cells large dense core vesicles still accumulate at the plasma membrane after BoNT/A treatment (Banerjee et al, 1996). In summary, the biochemical work, together with the morphological studies, indicates that vesicles can dock despite impaired or absent SNAREs, suggesting that synaptobrevin, syntaxin, and SNAP-25 are involved in events that occur between docking and fusion rather than being directly responsible for vesicle docking.…”

Syndet, an Adipocyte Target SNARE Involved in the Insulin-induced Translocation of GLUT4 to the Cell Surface

“…Higher order SDS-resistant ternary SNARE complexes have already been described in vitro (31). Interestingly, several groups have also implicated a role for the C terminus of SNAP-25 in a post-docking, fusion-catalyzing reaction (41)(42)(43). Some evidence exists to suggest the last 20 amino acids of this protein may play a role in Rab binding (44) and/or in activation of lipid-modifying reactions (45).…”