In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.

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.