Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases |
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Authors: | Wan Chen Kiessling Volker Cafiso David S Tamm Lukas K |
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Institution: | Center for Membrane Biology, Department of Molecular Physiology and Biological Physics, University of Virginia, Snyder Building, P.O. Box 800886, Charlottesville, Virginia 22908, United States. |
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Abstract: | Synaptotagmin I is the calcium sensor in synchronous neurotransmitter release caused by fusion of synaptic vesicles with the presynaptic membrane in neurons. Synaptotagmin I interacts with acidic phospholipids, but also with soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), at various stages in presynaptic membrane fusion. Because SNAREs can be organized into small cholesterol-dependent clusters in membranes, it is important to determine whether the C2 domains of synaptotagmin target membrane domains with different cholesterol contents. To address this question, we used a previously developed asymmetric two-phase lipid bilayer system to investigate the membrane binding and lipid phase targeting of soluble C2A and C2AB domains of synaptotagmin. We found that both domains target more disordered cholesterol-poor domains better than highly ordered cholesterol-rich domains. The selectivity is greatest (~3-fold) for C2A binding to disordered domains that are formed in the presence of 5 mol % PIP(2) and 15 mol % PS. It is smallest (~1.4-fold) for C2AB binding to disordered domains that are formed in the presence of 40 mol % PS. In the course of these experiments, we also found that C2A domains in the presence of Ca(2+) and C2AB domains in the absence of Ca(2+) are quite reliable reporters of the acidic lipid distribution between ordered and disordered lipid phases. Accordingly, PS prefers the liquid-disordered phase over the liquid-ordered phase by ~2-fold, but PIP(2) has an up to 3-fold preference for the liquid-disordered phase. |
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