As noted previously, shortly after a vesicle buds off from the donor membrane, the vesicle coat disassembles to uncover a vesicle-specific membrane protein, a v-SNARE (see Figure 14-6b). Likewise, each type of target membrane in a cell contains t-SNARE membrane proteins, which interact specifically with v-SNAREs. After Rab-mediated docking of a vesicle on its target membrane, the interaction of cognate SNAREs brings the two membranes close enough together that they can fuse.

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One of the best-understood examples of SNARE-mediated fusion occurs during exocytosis of secreted proteins (see Figure 14-10a, steps 2 and 3). In this case, the v-SNARE, known as VAMP (vesicle-associated membrane protein), is incorporated into secretory vesicles as they bud from the trans-Golgi network. The t-SNAREs are syntaxin, an integral membrane protein in the plasma membrane, and SNAP-25, which is attached to the plasma membrane by a hydrophobic lipid anchor in the middle of the protein. The cytosolic region in each of these three SNARE proteins contains a repeating heptad sequence that allows four α helices—one from VAMP, one from syntaxin, and two from SNAP-25—to coil around one another to form a four-helix bundle (Figure 14-10b). The unusual stability of this bundled SNARE complex is conferred by the arrangement of hydrophobic and charged amino acid residues in the heptad repeats. The hydrophobic amino acids are buried in the central core of the bundle, and amino acids of opposite charge are aligned to form favorable electrostatic interactions between helices. As multiple four-helix bundles form, the embedded transmembrane domains of VAMP and syntaxin pull the vesicle and target membranes together into very close apposition. The energetically favorable formation of four-helix bundles can overcome the electrostatic repulsion of the generally negatively charged phospholipid head groups in the vesicle and target membranes, allowing the hydrophobic interiors of the two membranes to come into contact, creating an opening between the two membranes, and ultimately causing the vesicle membrane to fuse with the target membrane.

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In vitro experiments have shown that when liposomes containing purified VAMP are incubated with other liposomes containing syntaxin and SNAP-25, the two classes of membranes fuse, albeit slowly. This finding is strong evidence that the close apposition of membranes resulting from formation of SNARE complexes is sufficient to bring about membrane fusion. Fusion of a vesicle and target membrane occurs more rapidly and efficiently in the cell than it does in liposome experiments in which fusion is catalyzed only by SNARE proteins. The likely explanation for this difference is that in the cell, other proteins, such as Rab proteins and their effectors, are involved in targeting vesicles to the correct membrane.

Yeast cells, like all eukaryotic cells, express more than 20 different related v-SNARE and t-SNARE proteins. Analyses of yeast mutants defective in each of the SNARE genes have identified specific membrane-fusion events 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 cis-Golgi network), each participating SNARE protein contributes only one α 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 could efficiently mediate 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, together with the specificity of interaction between Rab and Rab effector proteins, the specificity of the interaction between SNARE proteins can account for most, if not all, of the specificity of fusion between a particular vesicle type and its target membrane.