Proteolysis of SNAP‐25 Isoforms by Botulinum Neurotoxin Types A, C, and E

222

183

Regulated neurotransmitter secretion is a specialized version of a general membrane fusion mechanism in which exocytotic fusion is strictly Ca 2ϩ -regulated. Studies of this process have yielded insights into universal mechanisms for intracellular membrane fusion and the identity of core components of the fusion machinery (1-3). VAMP, 1 syntaxin, and SNAP25 are the neural protein substrates for clostridial neurotoxins, a family of highly selective proteases that potently inhibits neurosecretion (4, 5). These proteins are soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) that mediate the membrane association of N-ethylmaleimide-sensitive factor, a protein required for membrane fusion (6). The SNARE proteins are capable of assembling into extremely stable heterotrimeric complexes (7-9) that consist of a four-helix bundle in parallel alignment (10 -13). A current hypothesis suggests that the formation of SNARE complexes in trans across apposing membranes promotes intimate bilayer interactions and provides the energy to drive membrane fusion (10,(13)(14)(15)(16) (1,18,22,23). The precise role of synaptotagmin and its Ca 2ϩ -dependent interactions with phospholipids or syntaxin in regulated neurosecretion remains to be determined.Studies of regulated exocytosis in membrane preparations from neuroendocrine cells demonstrated that SNAREs are required for a late Ca 2ϩ -dependent step that occurs after vesicle docking and ATP-dependent priming and immediately before fusion (28,29). Tetanus toxin and botulinum neurotoxin (BoNT) B, C1, and E completely inhibited the Ca 2ϩ -dependent triggering of exocytosis, which implied that VAMP, syntaxin, and SNAP25 participate in steps close to or at fusion. Paradoxically BoNT A, which like BoNT E cleaves SNAP25, was only partially inhibitory for triggered fusion despite efficient proteolysis of SNAP25 (28). Because these toxins cleave SNAP25 at distinct C-terminal sites (Arg 180 -Ile 181 for BoNT E and Gln 197 -Arg 198 for BoNT A; see Refs. 30 and 31), it was inferred that the domain within the C terminus of SNAP25 between the toxin cleavage sites plays a distinct role in Ca 2ϩ -dependent membrane fusion events (28). In the present study, this domain of SNAP25 is revealed to be essential for Ca 2ϩ -dependent interactions with synaptotagmin. This finding clarifies classical observations that BoNT A inhibition of neurosecretion is partially reversed by elevating neuronal Ca 2ϩ levels (32). Moreover, the results suggest that an important role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca 2ϩ -dependent interactions between synaptotagmin and SNARE protein complexes. EXPERIMENTAL PROCEDURES Assays for Ca2ϩ -activated Exocytosis-PC12 cells grown in Dulbecco's modified culture medium supplemented with 5% horse serum and * This work was supported in part by National Institutes of Health Research Grants DK25861 and DK40428 (to T. F. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This ar...

Section: Assays For Camentioning

confidence: 75%

The C Terminus of SNAP25 Is Essential for Ca2+-dependent Binding of Synaptotagmin to SNARE Complexes

Gerona

Larsen²,

Kowalchyk

et al. 2000

222

183

“…Just before use, aliquots of the prenylated protein were loaded with the nonhydrolyzable nucleotide guanosine 5Ј-O-(3-thiotriphosphate)(GTP␥S) as described (19). The light chain of BoNT/E was expressed and purified as previously described (24).…”

Section: Methodsmentioning

confidence: 99%

The Intraacrosomal Calcium Pool Plays a Direct Role in Acrosomal Exocytosis

Blas¹,

Michaut²,

Treviño³

et al. 2002

119

125

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP 3 )-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP 3 -sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP 3 -sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.The acrosome reaction is an exocytotic process induced physiologically by the activation of sperm receptors by ligands in the zona pellucida of the oocyte. This interaction initiates a complex transduction mechanism leading to multiple fusions between the outer acrosomal membrane and the overlying plasmalemma resulting in the release of the acrosomal content and exposure of the molecules present on the inner acrosomal membrane.As in other regulated secretory events, calcium plays a central role in acrosome reaction (1). Recent results from different laboratories suggest that a first transient cytosolic calcium increase (probably mediated by T-type calcium channels (2)) leads to a second sustained increase of cytosolic calcium that is necessary for the acrosome reaction (3-9). Although the connection between the two events is not completely clear, the current hypothesis is that the first calcium increase causes the activation of a phospholipase C (PLC).1 There are several isoforms of PLC in the spermatozoa (10, 11). PLC␦4, in particular, has been implicated in the early events of the acrosome reaction (12). Active PLC would produce inositol 1,4,5-trisphosphate (IP 3 ) that would open IP 3 -sensitive calcium channels in the membrane of intracellular stores. The emptying of these stores would trigger the opening of store-operated calcium (SOC) channels in the plasma membrane causing a second and sustained calcium increase that would trigger acrosomal exocytosis (1, 7). Although direct proof for the nature of the calcium stores involved in this mechanism is lacking, severa...

“…Although SNAP-25 expression is confined almost exclusively to the brain and pancreatic ␤-cells, SNAP-23 is ubiquitously expressed in most tissues (24,26). Further, unlike SNAP-25, human SNAP-23 is insensitive to cleavage by BotA (27). To resolve the uncertainties surrounding the mechanism of the BotA-induced inhibition of SOCs in non-neuronal cells, we initially defined the SNAREs expressed in HEK 293 and COS-1 cells.…”

Section: Snap-25 Snap-23 and The Activation Of Socs-althoughmentioning

confidence: 99%

Activation of Store-operated Calcium Channels

Scott

Furuya

Trimble

et al. 2003

Store-operated calcium channels (SOC) play a central role in cellular calcium homeostasis. Although it is well established that SOC are activated by depletion of the endoplasmic reticulum calcium stores, the molecular mechanism underlying this effect remains ill defined. It has been suggested that SOC activation requires fusion of endomembrane vesicles with the plasmalemma. In this model, SNARE-dependent exocytosis is proposed to deliver channels or their activators to the surface membrane to initiate calcium influx. To test this hypothesis, we studied the requirement for membrane fusion events in SOC activation, using a variety of dominant-negative constructs and toxins that interfere with SNARE function. Botulinum neurotoxin A (BotA), which cleaves SNAP-25, did not prevent SOC activation. Moreover, SNAP-25 was not detectable in the cells where BotA was reported earlier to inhibit SOC. Instead, the BotA-insensitive SNAP-23 was present. Impairment of VAMP function was similarly without effect on SOC opening. We also tested the role of N-ethylmaleimide-sensitive factor, a global regulator of SNARE-mediated membrane fusion. Expression of a mutated N-ethylmaleimide-sensitive factor construct inhibited all aspects of membrane traffic tested, including recycling of transferrin receptors to the plasma membrane, fusion of endosomes with lysosomes, and retrograde traffic to the Golgi complex. Despite this global inhibition of vesicular fusion, which was accompanied by gross alterations in cell morphology, SOC activation persisted. These observations cannot be easily reconciled with the vesicle-mediated coupling hypothesis of SOC activation. Our findings imply that the SOC and the machinery necessary to activate them exist in the plasma membrane or are associated with it prior to activation.