SNARE Proteins

Proteins, lipids, and other biomolecules are transferred among different cellular compartments by transport vesicles, which carry hydrophilic cargoes in the aqueous lumen or hydrophobic ones in the lipid membrane. Crucially, each vesicle must pinch off from the membrane of its parent compartment and recognize and fuse with that of its target compartment in a highly specific and tightly regulated way, such as to release its contents at the right place at the right time (Bennett 1995). In addition, very high activation energy barriers must be overcome during fusion of the vesicle membrane with the target membrane (Chernomordik and Kozlov 2005). In all eukaryotic cells, these tasks are accomplished by a family of proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Sollner et al. 1993).

Although SNAREs differ substantially from one another with respect to size, structure, and cellular localization, they all have in common a conserved cytosolic coiled-coil domain of 60-70 residues known as the SNARE motif and a membrane-anchoring domain, such as a transmembrane sequence or cysteine-linked palmitoyl chains. SNARE proteins may be classified according to two different criteria: Traditionally, vesicle SNAREs (v-SNAREs) are distinguished from target-membrane SNAREs (t-SNAREs) because they are initially attached to different membranes. A more recent classification based on structural features (Fasshauer et al. 1998) distinguishes among four conserved subfamilies called R-, Qa-, Qb-, and Qc-SNARES, depending on whether a polypeptide chain contributes an arginine or a glutamine residue to a central region of the coiled coil termed the zero ionic layer (Sutton et al. 1998). By virtue of their SNARE motifs, and upon stimulation by a specific trigger, cognate v- and t-SNARE proteins avidly but reversibly assemble into a four-stranded coiled coil known as the SNAREpin, in which each of the above subfamilies is represented by one helix. Since the constituent protein subunits remain anchored to the membranes of different compartments or organelles, this assembly is said to be a trans-SNARE complex. Importantly, coiled-coil interactions first occur at the membrane-distal N-terminal end of the SNARE motif and then propagate towards the C-terminal membrane-binding domains, thus bringing the two membranes in closer contact. This zippering of the coiled-coil domain is energetically favorable and is thought to contribute substantially to surmounting the high activation energy barrier associated with membrane fusion (Weber et al. 1998). After fusion, all polypeptide chains reside in the same membrane, and the assembly is henceforth referred to as a cis-SNARE complex.

A very important and outstandingly well-studied SNARE protein complex is that mediating the Ca2+-triggered fusion of acetylcholine-transporting neuronal vesicles with the presynaptic plasma membrane, thereby releasing the neurotrans-mitter into the synaptic cleft (Hanson et al. 1997). Here, synaptobrevin (also known as vesicle-associated membrane protein, VAMP) acts as v-SNARE and R-SNARE, residing in the membrane of presynaptic vesicles and contributing an arginine residue to the zero ionic layer. By contrast, syntaxin 1 and SNAP 25 (soluble A-ethylmaleimide-sensitive factor attachment protein of 25 kDa) assume the roles of t-SNAREs and Q-SNAREs, both being attached to the presynaptic plasma membrane and carrying glutamine residues in the center of their coiled-coil regions. Synaptobrevin and syntaxin 1 each contribute one helix to the coiled coil of the SNARE complex and are anchored to their respective membranes through their C-terminal transmembrane segments, whereas SNAP 25 participates with two helices in the complex and is bound to the presynaptic plasma membrane through palmitoyl chains linked to cysteine residues in between the two coiled-coil segments.

The structure of the trimeric core SNARE complex has been solved by X-ray crystallography (Sutton et al. 1998). The hydrophobic interface of the four-stranded coiled-coil region is commonly grouped into layers, each of which consists of four residues, one from each constituent helix. In the center of this elongated assembly, the zero ionic layer, containing one arginine (R56 from synaptobrevin) and three glutamine residues (Q226 from syntaxin 1, Q53 and Q174 from SNAP 25), is flanked by two extended leucine-zipper regions whose layers are denoted +1, +2, etc. and -1, -2, etc., respectively. These coiled coils shield the zero ionic layer from water and thus enhance electrostatic attraction between the guanidinium group of the arginine and the carbonyl groups of the three glutamines. Without this stabilizing effect, the SNARE complex readily dissociates, which has been suggested to allow for recycling of SNARE proteins once membrane fusion is completed (Sutton et al. 1998).

Complex assembly upon neuronal stimulation by Ca2+ and disassembly upon acetylcholine release are controlled by an elaborate, but as yet poorly defined, network of SNARE master proteins. For instance, the vesicle-associated protein synaptotagmin acts as Ca2+ sensor and binds to certain membrane lipids as well as SNARE proteins, thereby modulating their oligomerization (Stein et al. 2007). Another regulatory mechanism is exerted by the N-terminal three-helix domain of syntaxin 1 itself, which can fold back onto the SNARE motif and thus prevent it from participating in coiled-coil assembly. Furthermore, this SNARE motif confers not only interaction with cognate SNARE proteins, but also binding to voltage-gated Ca2+ and K+ channels in the presynaptic plasma membrane (Bezprozvanny et al. 1995) and self-association of syntaxin 1 into homomeric protein clusters comprising about 75 monomers (Sieber et al. 2006, 2007). Clustering is believed to be essential for providing the high syntaxin 1 densities required for vesicle docking and fusion, but a detailed understanding of this phenomenon and the mode of action of SNARE masters is just beginning to emerge. Nevertheless, neuronal v- and t-SNAREs can be reconstituted separately into artificial lipid bilayers and spontaneously trigger vesicle fusion (Schuette et al. 2004). Very recently, the energetics and dynamics of SNAREpin formation have been assessed by measuring the forces exerted between v- and t-SNAREs embedded in two opposing, solid-supported lipid bilayers (Li et al. 2007), suggesting that assembly of a single SNARE coiled coil can release sufficient energy to fuel hemifusion, but not complete fusion, of the membranes.

In summary, coiled-coil-dependent SNARE complexes fulfill multiple key functions in vesicular trafficking, accounting for target-membrane recognition, vesicle tethering, and energy supply. Moreover, SNAREs are likely to serve as scaffolds for the recruitment and assembly of other proteins involved in docking and fusion and could thus be crucial for integrating these two processes (Gillingham and Munro 2003).

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