Vesicle docking and fusion

Petrucci TC, Macioce P, Paggi P (1991) Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells. J Neurosci 11 2938 16 Pevsner J, Scheller RH (1994) Mechanisms of vesicle docking and fusion insights from the nervous system. Curr Opin Cell Biol 6 555-60... 

The light chain, when separated from the heavy chain, behaves as an enzyme that selectively cleaves a peptide associated with synaptic vesicles. This peptide, synapt-obrevin, is required for docking and fusion of the vesicle during acetylcholine release. By enzymatically cleaving it, botulinum toxin renders vesicle docking and fusion impossible, and cholinergic neurotransmission comes to a halt. Because the light chain has enzymatic activity, just a few molecules can catalyze the destruction of synaptobrevin on thousands of vesicles. In this way, extreme potency is achieved via amplification of the process. 

Sacher, M., Jiang, Y., Barrowman, J., Scarpa, A., Burston, J., Zhang, L., Schieltz, D., Yates, J. R., 3rd, Abeliovich, H., and Ferro-Novick, S. (1998). TRAPP, a highly conserved novel complex on the aVGolgi that mediates vesicle docking and fusion. EMBO J. 17, 2494-2503. 

That impulse-evoked release of neurotransmitters depends on a Ca +-dependent extrusion from storage vesicles is beyond dispute. However, many details concerning the supply of vesicles that participate in this process, as well as the processes which regulate the docking and fusion of synaptic vesicles with the axolemma, remain uncertain. Nevertheless, it is clear that the amount of transmitter that is released in this... 

Calakos, N and Scheller, RH (1996) Synaptic vesicle biogenesis, docking and fusion a molecular description. Physiol. Rev. 76 1-29. 

Rab and Ral proteins Rab3A, Rab3C, Rab5, Rab7 and Ral. Since Rab proteins cycle between cytosolic and membrane-bound forms, not all synaptic vesicles contain all Rab proteins at the same time. Rab proteins regulate docking and fusion processes. 

Subtypes of Rab, particularly Rab3, have been implicated in the regulation of exocytosis and neurotransmitter release at nerve terminals (see also Chs 9 and 10) [30,31]. One possible scheme by which this might occur is shown in Figure 19-5. In its GTP-bound form, Rab associates with synaptic vesicles and interacts with other membrane proteins to create a complex unfavorable for vesicle docking and perhaps fusion. Upon depolarization of the nerve terminal, a Rab GAP is activated, which results in dissociation of the GDP form of Rab from the vesicle membrane. This enables the synaptic vesicle to proceed with... 

Neuromuscular transmission involves the events leading from the liberation of acetylcholine (ACh) at the motor nerve terminal to the generation of end plate currents (EPCs) at the postjunctional site. Release of ACh is initiated by membrane depolarization and influx of Ca++ at the nerve terminal (Fig. 28.1). This leads to a complex process involving docking and fusion of synaptic vesicles with active sites at the presynaptic membrane. Because ACh is released by exocytosis, functional transmitter release takes place in a quantal fashion. Each quantum corresponds to the contents of one synaptic vesicle (about 10,000 ACh molecules), and about 200 quanta are released with each nerve action potential. 

In eukaryotes, soluble N-ethylmaleimide-sensitive factor (NSF) adaptor proteins (SNAPs) receptors (SNAREs) are known to be required for docking and fusion of intracellular transport vesicles with acceptor/target membranes. The fusion of vesicles in the secretory pathway involves target-SNAREs (t-SNAREs) on the target membrane and vesicle-SNAREs (v-SNAREs) on vesicle membranes that recognize each other and assemble into trans-SNARE complexes (Sollner et al., 1993). 

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). 

In neurons, the SNARE complex consists of three main proteins the v-SNARE synaptobrevin or VAMP (vesicle-associated membrane protein), and two t-SNAREs, syntaxin and SNAP-25 (synaptosomal associated protein of 25 kD). Synaptobrevins traverse the synaptic vesicle membrane in an asymmetric manner a few amino acids are found inside the vesicle, but most of the molecule lies outside the vesicle, within the cytoplasm. Synaptobrevin makes contact with another protein anchored to the plasma membrane of the presynaptic neuron, syntaxin, which is associated with SNAP-25. Via these interactions, the SNARE proteins play a role in the docking and fusion of synaptic vesicles to the active zone. 

Figure 1 Overview of the synaptic vesicle cycle, (a) Within the presynaptic terminal, synaptic vesicles are filled with neurotransmitter by the action of specific vesicular neurotransmitter transporters, (b) Neurotransmitter-filled vesicles translocate to the active-zone membrane where they undergo docking, (c) Docked vesicles transition to a release-competent state through a series of priming or prefusion reactions, (d) Invasion of an action potential into the presynaptic terminal and subsequent calcium influx induces rapid fusion of the synaptic vesicle membrane with the terminal membrane, which thereby releases the neurotransmitter into the synaptic cleft, (e) Spent vesicles are internalized by clathrin-mediated endocytosis and are recycled for reuse, which thus completes the synaptic vesicle cycle. SV, synaptic vesicle CCV, clathrin-coated vesicle EE, early endosome. NOTE The use of arrows indicates a temporal sequence of events. Physical translocation of synaptic vesicles is unlikely to occur between the docking and fusion steps.

Rab proteins exist in all cells and form the largest branch of the Ras superfamily. This family performs a central function in vesicular transport. Rab proteins influence and regulate the budding, targeting, docking and fusion of vesicles as well as processes of exocytosis and endocytosis involving clathrin-coated vesicles. During these functions, Rab proteins cycle between the cytosol and the cell membrane, and this cyle is superimposed on a GDP/GTP cycle. The cytosolic pool of the Rab pro-... 

Synaptic vesicles are rapidly regenerated by endoc3rt Ic budding of clathrin-coated vesicles from the plasma membrane, a process that requires dynamin. After the clathrin coat Is shed, vesicles are refilled with neurotransmltter and move to the active zone for another round of docking and fusion. 

In conformity with the sequential processing of bacterial protein toxins such as diphtheria or cholera toxin, the action of BoNT involves multiple discrete steps binding to surface receptors, internalization via receptor-mediated endocytosis, transport from endosome to cytosol, and cleavage of target proteins in the cytosol. " Binding and internalization are mediated by the C- and N-terminal domains of the BoNT H-chain, respectively. The L-chains have zinc metalloprotease activity, targeted selectively to one of three proteins that are required for the docking and fusion of synaptic vesicles with active zones at the cytoplasmic surface of the nerve terminal. 

The toxin is a 150-kDa zinc-dependent metalloproteinase that cleaves proteins involved in the docking and fusion of synaptic vesicles to the membrane at the neuromuscular junction. The toxin consists of two chains a heavy chain (100 kDa) and a light chain (50kDa) linked by a disulfide bond. The crystal structure of the molecule has been solved to 3.3-A resolution [49]. The protein structure revealed a 50-residue belt that partially obscures the active site access channel the authors note that this unusual feature makes rational inhibitor design more difficult. 

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