Abstract
The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial lipid-binding Protein (SMP) domain, a lipid-harboring module through which they mediate lipid transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and lipid transport functions of E-Syt1. Its membrane tethering activity is monitored through a turbidity-based assay, and its lipid transport property is analyzed via fluorescence resonance energy transfer (FRET)-based assay. These in vitro methods have enabled us to gain insight into the mechanism of action and regulation of E-Syt1, such as the role of Ca2+ in releasing E-Syt1 from an autoinhibitory conformation. The same methods could be adapted to the study of other lipid transport proteins that function at membrane contact sites.
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Acknowledgments
We thank Frederic Pincet, Joshua Lees, Frank Wilson, Heather Wheeler, Louise Lucast, and Yiying Cai for technical assistance. P.D.C. was supported in part by NIH grants NS036251 and DA018343. X.B. was supported by a Human Frontier Science Program long-term fellowship.
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Bian, X., De Camilli, P. (2019). In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_15
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DOI: https://doi.org/10.1007/978-1-4939-9136-5_15
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