Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers

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Abstract
Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER–PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.
Funding Information
  • U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (R01NS113236, R37NS036251)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35GM131714, R01GM093341, R01GM120193)

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