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SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate. [ABSTRACT FROM AUTHOR]

SNARE complex formation is essential for intracellular membrane fusion. Vesicle-associated (v-) SNARE intertwines with target membrane (t-) SNARE to form a coiled coil that bridges two membranes and facilitates fusion. For the SNARE family involved in neuronal communications, complex formation is tightly regulated by the v-SNARE-membrane interactions. However, it was found using EPR that complex formation is spontaneous for a different SNARE family that is involved in protein trafficking in yeast. Further, reconstituted yeast SNAREs promoted membrane fusion, different from the inhibited fusion for reconstituted neuronal SNAREs. The EPR structural analysis showed that none of the coiled-coil residues of yeast v-SNARE is buried in the hydrophobic layer of the membrane, making the entire coiled-coil motif accessible, again different from the deep insertion of the membrane-proximal region of neuronal v-SNARE into the bilayer. Importantly, yeast membrane fusion is constitutively active, while synaptic membrane fusion is regulated, consistent with the present results for two SNARE families. Thus, the v-SNARE-membrane interaction may be a major molecular determinant for regulated versus constitutive membrane fusion in cells. [ABSTRACT FROM AUTHOR]