SNARE hypothesis

Protein transport between intracellular compartments is mediated by a mechanism that is well-conserved among all eukaryotes, from yeast to man. The transport mechanism involves carrier vesicles that bud from one organelle and fuse selectively to another. Specialized proteins are required for vesicle transport, docking, and fusion, and they have been generically named SNAREs (an acronym for soluble N-ethylma-leimide-sensitive fusion attachment protein receptor). SNAREs have been divided into those associated with the vesicle (termed v-SNAREs), and those associated with the target (termed t-SNAREs). The key protein, which led to the discovery of SNAREs was NSF, an ATPase found ubiquitously in all cells, and involved in numerous intracellular transport events. The subsequent identification of soluble proteins stably bound to NSF, the so-called SNARE complex, led to the formulation of the SNARE hypothesis, which posits that all intracellular fusion events are mediated by SNAREs (Rothman, 2002). 

Serotypes B, D, E, and G cleave different sites on the synaptic vesicle protein, synaptobrevin (VAMP), whereas serotypes A and E cleave the presynaptic membrane-associated protein SNAP-25 (Schiavo et al., 2000 Simpson, 2004). Serotype Cl is unique in that it cleaves two cytoplasmic proteins, syntaxin and SNAP-25 (Wiltiamson et al., 1996). Interaction of these SNAREs on the surface of synaptic vesicles and active zone membranes is required for voltage- and Ca " -dependent release of neurotransmitter cleavage by BoNT inhibits this process, leading to muscle weakness and paralysis (Sutton et al., 1998 Schiavo et al., 2000). Cleavage of SNARE proteins appears to be sufficient to account for all actions of the BoNTs, and the SNARE hypothesis has received near universal acceptance since its introduction in the early 1990s. 

BoNT inhibits this process, leading to mnscle weakness and paralysis. Cleavage of SNARE proteins appears to be sufficient to account for aU actions of the BoNT. The SNARE hypothesis has received near-universal acceptance since its introduction in the early 1990s an alternative hypothesis has been advanced based on results obtained in PC12 pheochromocytoma cells,however, its generality has not yet been established. 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

When membranes fuse, the so-called stalk hypothesis suggests that the intermediate hemifusion state (Fig. 6.4c) comprises a structure in which proximal monolayers layers are connected by a bent stalk and the distal layers are pulled towards each other, thus forming a dimple (see also Fig. 6.5) The stalk model has been supported by theoretical and experimental observations. The fusion of model membranes appears to occur via the same series of fusion intermediates as those in vivo, although the approach of membranes is not Rab/SNARE mediated but is driven by reduced bilayer repulsion forces arising from hydration, electrostatic interactions, thermal fluctuations (Helfrich interaction) or osmotic stress. Membrane fusion is also promoted by defects introduced into the membrane by lateral phase separation (for example of lipid rafts, see above), high spontaneous membrane curvature, or addition of macromolecules or proteins into the membrane. 

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