title: Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases.
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.
authors.affiliation: 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.
authors.forename: Chen
authors.initials: C
authors.lastname: Wan
authors.forename: Volker
authors.initials: V
authors.lastname: Kiessling
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
authors.forename: Lukas K
authors.initials: LK
authors.lastname: Tamm
mesh_terms: D000818:Animals; D002118:Calcium; D002784:Cholesterol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011806:Quinolinium Compounds / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: C481052:Alexa fluor 546; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D050505:Mutant Proteins; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; D011806:Quinolinium Compounds; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D053835:Unilamellar Liposomes; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D002784:Cholesterol; D002118:Calcium