T-SNARE proteins

A second mechanism that impinges on the localization of transporters is through the association with proteins, the most prominent example being syntaxin. Syntaxin is a t-SNARE protein necessary for the fusion of vesicles with the plasma membrane (see the chapter on exocytosis). On the cell surface syntaxin consistently stabilizes the localization of GABA, noradrenaline, glycine, and 5HT transporters the PKCa isoform can sever the interaction with syntaxin suggesting a general mechanism for transporter internalization. 

Based largely on a proposal by Rothman and colleagues, anterograde vesicular transport can be considered to occur in eight steps (Figure 46-7). The basic concept is that each transport vesicle bears a unique address marker consisting of one or more v-SNARE proteins, while each target membrane bears one or more complementary t-SNARE proteins with which the former interact specifically. 

Yeast cells, like all eukaryotic cells, express more than 20 different related v-SNARE and t-SNARE proteins. Analyses of yeast sec mutants defective in each of the SNARE genes have identified the specific membrane-fusion event in which each SNARE protein participates. For all fusion events that have been examined, the SNAREs form four-helix bundled complexes, similar to the VAMP/syntaxin/SNAP-25 complexes that mediate fusion of secretory vesicles with the plasma membrane. However, in other fusion events (e.g., fusion of COPII vesicles with the c7s-Golgi network), each participating SNARE protein contributes only one a helix to the bundle (unlike SNAP-25, which contributes two helices) in these cases the SNARE complexes comprise one v-SNARE and three t-SNARE molecules. 

Using the in vitro liposome fusion assay, researchers have tested the ability of various combinations of individual v-SNARE and t-SNARE proteins to mediate fusion of donor and target membranes. Of the very large number of different combinations tested, only a small number mediated membrane fusion. To a remarkable degree the functional combinations of v-SNAREs and t-SNAREs revealed in these in vitro experiments correspond to the actual SNARE protein interactions that mediate known membrane-fusion events in the yeast cell. Thus the specificity of the interaction between SNARE proteins can account for the specificity of fusion between a particular vesicle and its target membranes. 

Each v-SNARE in a vesicular membrane specifically binds to a complex of cognate t-SNARE proteins in the target membrane. Inducing fusion of the two membranes. After fusion is completed, the SNARE complex is disassembled in an ATP-dependent reaction mediated by other cytosolic proteins (see Figure 17-11). 

FIGURE 5.19 Role of cholesterol in the exocytosis of synaptic vesicles. Cholesterol constrains the conformation of v-SNARE proteins in such a way that their transmembrane domains adopt a parallel ("closed scissors") orientation. This particular conformation is required for the fusion process because it induces a fusion-compatible curvatiue of the s)fnaptic vesicle. In this case, the interaction between v-SNARE and t-SNARE proteins triggers the fusion mechanism (lower inset, 2). Without cholesterol, v-SNARE remains in an "open scissors" conformation (upper inset, 1), and the curvature of the vesicle is not appropriate for fusion. ... 

Seel A member of a family of proteins that attach to t-SNAREs and are displaced from them by Rab proteins, thereby allowing v-SNARE-t-SNARE interactions to occur. 

Step 5 Vesicle targeting is achieved via members of a family of integral proteins, termed v-SNAREs, that tag the vesicle during its budding. v-SNAREs pair with cognate t-SNAREs in the target membrane to dock the vesicle. 

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Araque A., Li N., Doyle R. T., and Haydon P. G. (2000). SNARE protein-dependent glutamate release from astrocytes. J. Neurosci. 20 666-673. 

Yokoyama CT, Myers SJ, Fu J, Mockus SM, Scheuer T, Catterall WA (2005) Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site. Mol Cell Neurosci 28 1-17 Yokoyama CT, Sheng ZH, Catterall WA (1997) Phosphorylation of the synaptic protein interaction site on N-type calcium channels inhibits interactions with SNARE proteins. J Neurosci 17 6929-38... 

Blackmer T, Larsen EC, Bartleson C et al (2005) G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nat Neurosci 8 421-5 Boczan J, Leenders AG, Sheng ZH (2004) Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis. J Biol Chem 279 18911-19... 

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. 

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