Fusion Step-Specific Influence of Cholesterol on SNARE-Mediated Membrane Fusion

Abstract

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. Using the fluorescence assay and dye-labeled SNAREs and the fluorescent lipids, 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-fold and three-fold for subsequent pore formation and pore expansion at 40 mol %, respectively. 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.

Transmembrane-domain determinants for SNARE-mediated membrane fusion

“…Severe secretory deficits are observed when shortening or lengthening the C-terminal, but not the Nterminal half of the TMD, suggesting that distinct structural length requirements within the VAMP2 TMD are largely confined to the C-terminal part. It is thus tempting to speculate that the conformation of the C-terminal part of the VAMP2 TMD, which can be further modulated by the lipid environment (Tong et al, 2009;Chang et al, 2009), might regulate the formation of a hemifusion state (Chernomordik and Kozlov, 2005;Xu et al, 2005;Hofmann et al, 2006;Wong et al, 2007;Lu et al, 2008;Liu et al, 2008;Chang et al, 2009) en route to full membrane merging.…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…A more recent mouse model carries the D1005G mutation in NPC1 ( 73 ). This mutation is in the cysteinerich loop of NPC1, the site at which the most common mutations occur in human NPC patients, and these mice have a more slowly progressing disease than that of NPC1-null by guest, on May 10, 2018 www.jlr.org Downloaded from addition, the formation of presynaptic SNARE complexes, the interaction between synaptobrevin and synaptophysin, the fusion of synaptic vesicles with presynaptic membranes, and the sorting of synaptic vesicles during endocytosis all depend on cholesterol (120)(121)(122)(123)(124). Thus, extraction of cholesterol from neurons, by brief incubation with cyclodextrin, impaired exocytosis and endocytosis of synaptic vesicles ( 114,(125)(126)(127)(128).…”

Effect of Hydration and Packing Order on Large Unilamellar Vesicle Fusion: The Role of Cholesterol

“…Recently, a series of studies have reported that cholesterol regulates the fusion process between vesicles and cells, and most of these papers report that these system mergers are associated with the formation of cholesterol-rich domains or "rafts" that favor the fusion process [21][22][23][24] . In a vesicle-vesicle PEG-dependent fusion process, cholesterol has been reported to induce negative curvature that generates instability in some systems and favors breaking over merger in some mixtures 19 .Cholesterol affects the physicochemical properties of the lamella by dramatically increasing the order of acyl chains and inducing regular distribution domains.…”