However, genetic and other perturbation experiments indicated that SNARE proteins were not required for docking. In the original SNARE hypothesis, contacts between the SNARE proteins were proposed to confer specificity during docking. It is recognized that protein interactions must specifically associate a vesicle to the correct target membrane. In addition, the molecular basis for docking is unknown. Thus, even the morphological definition of docked vesicles varies in the literature. Moreover, because standard fixation methods often introduce changes in membrane structure, docking is sometimes defined as including all vesicles near the membrane-usually specified as vesicles within about 30 nm of the membrane. However, the precise definition of docking is a muddle since morphologically docked vesicles are thought to include those in both the primed and unprimed pools. Synaptic vesicle docking is observed in electron micrographs of the synapse and is defined as the attachment of vesicles to their target membranes. Äocking precedes priming and at this point is defined solely by morphological criteria. These primed vesicles are likely to correspond to the physiologically defined readily releasable pool. Thus, the SNARE proteins function both in priming and in fusion. It is believed that the SNARE proteins partially wind into a complex, but membrane fusion is arrested, and the vesicle is held in this state until triggered to fuse by an increase in calcium. Priming describes a molecular state in which a four-helix SNARE complex has formed between SNARE proteins on a synaptic vesicle and those on the plasma membrane. The formation of this tightly wound structure may provide the driving force for fusion. For synaptic vesicle fusion, the vesicular SNARE protein synaptobrevin (also called vesicle-associated membrane protein or VAMP) interacts with the plasma membrane SNARE proteins syntaxin and SNAP-25 to form a four-helix bundle. Specific sets of complementary SNARE proteins are localized to each cargo vesicle and target compartment in the cell and thereby provide dedicated fusion proteins for each trafficking event. When reconstituted into liposomes under permissive conditions, the SNARE proteins have been demonstrated to be necessary and sufficient for membrane fusion. The molecular basis of fusion is thought to be mediated by the soluble N-ethylmaleimide–sensitive fusion attachment protein receptors SNARE proteins. Fusing vesicles can be observed by electron microscopy and by electrophysiological recordings. For example, the final step of vesicle fusion, in which vesicles fuse with the plasma membrane, is well defined by these three criteria. A goal of studies in neurotransmission is to define the state of the vesicle at each step in exocytosis using morphological, physiological, and molecular criteria. The biological state of a synaptic vesicle can be defined by three distinct parameters: morphology (its location in the synapse) physiology (its release competence) and molecular interactions. Fusion of synaptic vesicles with the plasma membrane is thought to occur in three ordered steps: docking, priming, and fusion.
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