Synaptotagmin-1 binds to PIP2-containing membrane but not to SNAREs at physiological ionic strength

Abstract

Ca2+-sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing PIP2 is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg2+ completely abrogate synaptotagmin-1 binding to SNAREs regardless of whether Ca2+ is present or not. Altogether, our data suggest that at physiological ion concentrations Ca2+-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium

“…To test this hypothesis, we measured InsP 3 binding to the isolated C2B domain in the presence of SNARE complex and in the absence of Ca 2+ (to avoid precipitation of protein [Dai et al, 2007]) by ITC. The presence or absence of SNARE-complex made no difference to InsP 3 binding to the isolated C2B domain (Figure 4f), in agreement with previous observations from our group (Park et al, 2015) . This evidence suggests that, under physiological conditions, the C2AB fragment binds preferentially to PtdIns(4,5)P 2 .…”

“…As discussed below, the relative affinity of the C2AB fragment for PtdIns(4,5)P 2 versus its affinity for the SNAREs suggests that PI(4,5)P 2 is the likely target of the polybasic patch of the C2B domain, in agreement with recent reports (Wang, 2016; Park et al, 2015). Furthermore, Ca 2+ increases C2B's affinity for PtdIns(4,5)P 2 , probably by neutralizing the negatively charged side chain at the Ca 2+ -binding site of the C2B domain, allowing a tighter binding of the polybasic patch to PtdIns(4,5)P 2 .…”

Reconstitution of SNARE Mediated Membrane Fusion

“…DCV fusion was SNARE-dependent in all conditions tested in the planar supported bilayer assay Table 8.2, consistent with results for other purified secretory vesicles preparations Park, et al, 2015). The amount of docking as well as the rate and probability of fusion of DCVs to supported bilayers were enhanced by calcium (Figure 8.3, Table 8.3, and Table 8.4).…”

“…Numerous studies have identified syaptotagmin as a calcium sensor that triggers fusion (Südhof, 2013;Brose, et al, 1992). Notably, synaptotagmin has been shown to accelerate fusion by interacting with PI4,5P 2 Park, et al, 2015;Wang, et al, 2011). Some of these effects could be due to increased crosslinking of vesicles by synaptotagmin although single particle fusion assays have indicated that synaptotagmin may have little effect on docking in the presence of SNARE proteins .…”

“…Fusion of physiological vesicles has been shown for purified synaptic vesicles (SV) from rat brains (Holt, et al, 2008;Park, et al, 2012;Kiessling, et al, 2013) and purified dense core vesicles (DCVs) from bovine chromaffin cells Park, et al, 2015). SV and DCVs exhibit accelerated lipid mixing in the presence of calcium (Holt, et al, 2008;Park, et al, 2012) and this was shown to be caused by an increase and acceleration of fusion when SV were observed in the single vesicle planar supported bilayer fusion assay .…”