Calcium drives fusion of SNARE‐apposed bilayers

Abstract

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.

Secretion machinery at the cell plasma membrane

“…These studies demonstrate that hydrated calcium [Ca(H 2 O) n ] 2+ has more than one shell around the Ca 2+ , with the first hydration shell having six water molecules in an octahedral arrangement [39]. In studies using light scattering and x-ray diffraction of SNARE-reconstituted liposomes, it has been demonstrated that fusion proceeds only when Ca 2+ ions are available between the t-and v-SNARE-apposed proteoliposomes [12,33].…”

“…The remaining option has been the use of nuclear magnetic resonance spectroscopy (NMR), however the NMR approach too has not been successful, primarily due to the molecular size limitation of the NMR. Regardless of these set backs and limitations, AFM force spectroscopy has provided for the first time at nm resolution, an understanding of the structure, assembly, and disassembly of the membrane-associated t-/v-SNARE complex in physiological buffer solution [7,12,17 …”

“…2F), to allow appropriate t-/v-SNARE interactions leading to membrane fusion in the presence of calcium [7]. Studies using SNARE-reconstituted liposomes and bilayers [12] demonstrate: (i) a low fusion rate (τ=16 min) between t-and v-SNARE-reconstituted liposomes in the absence of Ca 2+ ; and (ii) exposure of t-/v-SNARE liposomes to Ca 2+ drives vesicle fusion on a near physiological relevant time-scale (τ ~10s), demonstrating Ca 2+ and SNAREs in combination to be the minimal fusion machinery in cells [12]. Native and synthetic vesicles exhibit a significant negative surface charge primarily due to the polar phosphate head groups.…”

“…These polar head groups produce a repulsive force, preventing aggregation and fusion of opposing vesicles. SNAREs bring opposing bilayers closer, to within a distance of 2-3 Å, [12] enabling Ca 2+ bridging. The bound Ca 2+ then leads to the expulsion of water between the bilayers at the bridging site, leading to lipid mixing and membrane fusion.…”

“…The establishment of continuity between secretory vesicle membrane and the porosome membrane requires calcium and the assembly of a molecular complex [7] formed when specific proteins [8][9][10][11] in opposing bilayers interact. At the nerve terminal for instance, target membrane proteins SNAP-25 and syntaxin (t-SNAREs) associated at the base of porosomes [4], and synaptic vesicle-associated protein VAMP or v-SNARE, interact in a circular array [7] to form conducting channels in the presence of calcium [7,12]. During cell secretion, swelling of secretory vesicles result in a build-up of intra-vesicular pressure, required for the expulsion of vesicular contents through porosomes to the outside [13].…”