Partitioning of Synaptotagmin I C2 Domains between Liquid-Ordered and Liquid-Disordered Inner Leaflet Lipid Phases

Ca2+-triggered membrane fusion of synaptic vesicles with the presynaptic plasma membrane of neurons is the central event in synaptic transmission. This process is tightly regulated by synaptotagmin I (syt I)1, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins and other factors (1-3). Assembly of the four helix bundle SNARE protein complex docks the vesicles to the presynaptic membrane and is believed to be the essential reaction that drives membrane fusion (4-8). Syt I is an integral membrane protein of synaptic vesicles and is the Ca2+ sensor for synchronous neurotransmitter release (9-13). It is anchored to the membrane by a single transmembrane domain near the N-terminus and contains two C2-type Ca2+-sensing domains (C2A and C2B) towards its C-terminal end (14). A long linker connects C2A to the TM domain and a short linker connects C2A and C2B. C2A and C2B have similar folds consisting of a β-sandwich formed from two 4-stranded β-sheets. The loops that connect the β-strands protruding from one end form binding sites for Ca2+ ions, binding three or two Ca2+ ions, respectively. Ca2+ bound to these loops mediates the binding of C2 domains to negatively charged membrane surfaces (15, 16). In general, syt I C2 domains can interact with the synaptic vesicle membrane (cis-membrane) as well as with the presynaptic plasma membrane (trans-membrane) (17). In the following we will only consider the trans-bilayer-interactions. The C2 domains of syt I have been proposed to regulate fusion through interaction with acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol (PI) and its phosphorylated derivatives by binding and penetrating into the presynaptic membrane surface upon Ca2+ binding (18-20). In fact, when expressed as independent domains, the two C2 domains have been reported to prefer different membrane compositions. The membrane binding of C2A and C2B are strongly Ca2+-dependent and they bind well to bilayers composed of negatively charged lipids. However, C2A has a preference for PC/PE and PS containing bilayers, while C2B prefers to bind to bilayers containing PIP2. C2B even binds weakly in the absence of Ca2+ to PC/PE/PS or PC/PE/PIP2 bilayers through a highly basic strand in its β-sandwich (21-23). When expressed as tandem C2A and C2B domains, the two domains reinforce each other's binding and membrane penetration behavior (24). Expressed C2A and C2B have also been reported to change membrane curvature (25) and full-length syt I co-reconstituted with SNAREs can enhance SNARE-mediated fusion under appropriate conditions (26, 27). However, exactly how syt I co-operates with SNAREs and thereby imparts Ca2+ control on neuronal exocytosis is not yet fully understood. The SNAREs syntaxin 1A and SNAP-25 concentrate in partially overlapping clusters in plasma membranes that are also the sites of exocytosis in neuroendocrine cells (28) and pancreatic β cells (29). Cholesterol depletion disperses these clusters in plasma membranes and simultaneously impairs exocytosis. Although these clusters are cholesterol-dependent, SNAREs are not found in detergent-resistant fractions and therefore, are unlikely targeted to “rafts” in cell membranes. These results raise the question of whether syt I C2 domains prefer cholesterol-rich or cholesterol-poor regions of the plasma membrane and whether regulation of such partitioning could regulate potential interactions with SNAREs. Since C2 domains target the cytoplasmic (inner) leaflets of plasma and intracellular membranes, one strategy to answer this question is to use model membranes with coexisting liquid-ordered (lo) and liquid-disordered (ld) domains and inner leaflet lipid compositions. Unlike mixtures of PC, SM, and cholesterol, typical inner leaflet lipid mixtures of PC, PE, PS and cholesterol do not form phase-separated lo and ld phases on their own in model membranes (30, 31). However, such domains can be induced to form by phase coupling adjacent to phase-separated lo-ld domains formed by outer leaflet lipid PC, SM, and cholesterol mixtures in asymmetric supported lipid bilayers (30, 32). In the current work, we investigate the Ca2+-dependent binding and surface distribution of syt I C2A and C2AB on asymmetric bilayers with induced coexisting lo-ld phases, as well as the Ca2+-independent binding of C2AB on these bilayers as a function of the concentrations of the anionic lipids PS and PIP2 and the curvature-inducing lipid PE in the inner leaflet. We find that under all lipid conditions, C2 domains favor ld over lo lipid phases in the presence and absence of Ca2+. C2A's binding preference for ld phases is independent of the PS concentration, probably reflecting a concentration-independent partitioning of this lipid between ordered and disordered phases. However, C2A partitioning between lo and ld lipid phases depends on the PIP2 concentration, indicating a progressively higher accumulation of PIP2 in disordered membrane regions as PIP2 and C2A levels are increased. The selectivity of C2AB for disordered membrane regions is not as high as that of C2A and, importantly, C2AB in the absence of Ca2+can be used as a reliable reporter of acidic lipid partitioning between inner leaflet ld and lo phases in asymmetric lipid bilayers.