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.

In neurons, synaptotagmin1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca 2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca 2+ -triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca 2+ -sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca 2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca 2+ levels, and may function as a dynamic presynaptic Ca 2+ sensor to control the probability of neurotransmitter release.

Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40–50% of cases are positive. To uncover other potential targetable mutations, we performed whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, D816) wildtype melanoma. Surprisingly, we found a somatic BRAF L597R mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF V600 mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that 2 (4%) harbored L597 mutations and another 2 involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by MEK inhibition. A patient with BRAF L597S mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data demonstrate clinical significance to BRAF L597 mutations in melanoma.