| Cholesterol is a major component of biological membranes and is known to affect vesicle fusion. However, the mechanism by which cholesterol modulates SNARE-dependent intracellular fusion is not well understood. First, using the fluorescence assay and employing dye-labeled SNAREs and the fluorescent lipids on yeast post golgi trafficking SNARE-mediated model membrane fusion, we dissected cholesterol effects on individual fusion steps including SNARE complex formation, hemifusion, pore formation, and pore dilation. At physiological high concentrations, cholesterol stimulated hemifusion as much as 30-fold, but its stimulatory effect diminished to 10- and 3-folds for subsequent pore formation and pore expansion at 40 mole%, respectively. Therefore, the results show that cholesterol serves as a strong stimulator for hemifusion but acts as mild stimulators for pore opening and expansion. Strong stimulation of hemifusion and mild stimulation of pore formation are consistent with the fusion model based on the intrinsic negative curvature of cholesterol. However, even a milder effect of cholesterol on pore expansion is contradictory to such a simple curvature-based prediction. Thus, we speculate that cholesterol also affects the conformation of the transmembrane domains of SNAREs, which modulates the fusion kinetics. Furthermore, we investigated the effect of cholesterol in the specific steps of fusion: docking, lipid mixing, spontaneous fusion, and Ca2+-triggered fusion using single vesicular study. Cholesterol enhances the kinetics of all these steps. Cholesterol on both sides of the vesicles is required for content mixing. In addition, cholesterol helped Syntaxins assemble to form higher oligomers meanwhile the higher oligomer ratio of Vamp was not affected by cholesterol content. alpha-Synuclein (alpha-Syn), a major component of Lewy bodies that are considered as a hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of alpha-Syn decreases the neurotransmitter release. However, the mechanism by which alpha-Syn inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of alpha-Syn on SNARE assembly and SNARE-dependent liposome fusion using fluorescence methods. The results show that alpha-Syn inhibits bulk lipid mixing. Furthermore, mutants linked to familial PD A30P, A53T, and E46K are less effective than, similar to, more than the wild-type in fusion-inhibition, respectively, correlating well with the rank order of their individual membrane affinities. A negatively-charged lipid that strongly favors alpha-Syn's membrane binding is also required for the fusion-inhibiting function. Finally, the single-vesicle fusion assay reveals that alpha-Syn specifically inhibits vesicle docking, without interfering with lipid mixing and pore opening. Thus, alpha-Syn may inhibit SNARE-dependent vesicle docking through membrane binding. |
