Domain Coupling in Asymmetric Lipid Bilayers and Targeting of Synaptotagmin I C2 Domains to Phase-Separated Model Membranes

Mammalian cell membranes are highly heterogeneously structured in terms of lipid composition and asymmetry, and the lateral organization of membrane proteins. Lipids and/or membrane proteins may cluster into functional units ("rafts" or l o phases in model membranes), which could play critical roles in important cellular processes, particularly in signal transduction and membrane trafficking. In model membranes, rafts formed only by the lipids typically found in the outer leaflets, but not those of the inner leaflets of plasma membranes. This raises a major unsolved question of the raft hypothesis of signal transduction, which is how signals transmit across the two leaflets of membranes. To solve this problem from a biophysical perspective, we established supported asymmetric lipid bilayer systems that exhibited coexisting liquid-ordered (l o ) and liquid-disordered (l d ) domains based on our group‟s finding of transbilayer domain coupling. We examined the effects of lipid composition on the lipid phase behavior with these systems. It appears that the phase behavior is dominated by the intrinsic chain melting temperatures of the lipids, rather than by their specific headgroup classes. This research significantly improves our understanding of the mechanisms of transbilayer domain coupling, which could be a membrane organizing principle for signal transduction and membranemediated regulation. Many proteins segregate into either "raft" or "non-raft" clusters in plasma membranes, including SNAREs that mediate a variety of membrane fusion processes. ii However, the localization of synaptotagmin I (calcium sensor in SNARE-mediated membrane fusion) on heterogeneous membranes has rarely been studied previously. Since SNAREs can be organized into small cholesterol-dependent clusters in membranes, it is important to determine whether the C2 domains of synaptotagmin I target membrane domains of different cholesterol content. To address this question, we investigated the membrane binding and lipid phase targeting of soluble C2A and C2AB domains of synaptotagmin I on asymmetric two-phase lipid bilayer systems. We found that both domains target more disordered cholesterol-poor domains better than highly ordered cholesterol-rich domains. This binding preference has been quantified under different inner leaflet lipid and buffer conditions. This investigation provides important new information about the co-localization of critical components of the neuronal exocytotic fusion machinery.

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