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 enabl

SUBMITTER: Bian X 

PROVIDER: S-EPMC6481592 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature