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.

Tricalbin proteins regulate plasma membrane phospholipid homeostasis

“…The loss of PM specific phospholipid species is consistent with a role of ER-PM contacts in coordinating both phospholipid synthesis and transfer. All E-Syt family members feature a SMP domain that dimerizes and transports glycerolipids in vitro (Lee & Hong, 2006;Toulmay & Prinz, 2012;Schauder et al, 2014;Saheki et al, 2016;Yu et al, 2016;Bian et al, 2018;Bian & De Camilli, 2019;Qian et al, 2021). In particular, a recent study demonstrated the ability of the Tcb3 SMP domain to transport phospholipids in vitro (Qian et al, 2021), consistent with our in vivo experiments.…”

“…The E-Syt proteins, as well as their budding yeast orthologs, named tricalbins, are anchored in the ER membrane via a N-terminal hairpin anchor (Giordano et al, 2013) and interact with the PM in a phosphoinositide lipid-and Ca 2+ -dependent manner via their multiple C-terminal cytoplasmic C2 domains (Chang et al, 2013;Giordano et al, 2013;Idevall-Hagren et al, 2015;Saheki et al, 2016;Bian et al, 2018). They also feature a central cytosolic synaptotagmin-like, mitochondrial (SMP) domain that dimerizes and contains a deep hydrophobic groove previously shown to bind and transport lipids in vitro (Schauder et al, 2014;Saheki et al, 2016;Yu et al, 2016;Bian et al, 2018;Bian & De Camilli, 2019). While cellular roles of E-Syt proteins as ER-PM tethers are described (Giordano et al, 2013;Fern ández-Busnadiego et al, 2015), roles of the E-Syts in membrane lipid dynamics in vivo are enigmatic.…”

Tricalbin proteins regulate plasma membrane phospholipid homeostasis

“…The specific loss of PM phospholipid species is consistent with a role of the Tcb proteins in coordinating phospholipid synthesis and transfer at ER-PM contacts. All E-Syt family members feature a SMP domain that dimerises and transports glycerolipids in vitro (Lee and Hong, 2006; Toulmay and Prinz, 2012; Schauder et al, 2014; Saheki et al, 2016; Yu et al, 2016; Bian et al, 2018; Bian and De Camilli, 2019; Qian et al, 2021). In particular, a recent study demonstrated the ability of the Tcb3 SMP domain to transport phospholipids in vitro (Qian et al, 2021), consistent with our in vivo experiments.…”

“…The E-Syt proteins, as well as their budding yeast orthologs, named tricalbins, are anchored in the ER membrane via a N-terminal hairpin (Giordano et al, 2013) and interact with the PM in a phosphoinositide lipid- and Ca 2+ -dependent manner via their multiple C-terminal cytoplasmic C2 domains (Chang et al, 2013; Giordano et al, 2013; Idevall-Hagren et al, 2015; Saheki et al, 2016; Bian et al, 2018). They also feature a central cytosolic synaptotagmin-like, mitochondrial (SMP) domain that dimerizes and contains a deep hydrophobic groove previously shown to bind and transport lipids in vitro (Schauder et al, 2014; Saheki et al, 2016; Yu et al, 2016; Bian et al, 2018; Bian and De Camilli, 2019; Qian et al, 2021). Whilst cellular roles of E-Syt proteins as ER-PM tethers are well-described (Giordano et al, 2013, Fernández-Busnadiego et al 2015), precise roles of the E-Syts the control of membrane lipid dynamics in vivo are enigmatic.…”

ER-PM Contact Sites – SNARING Actors in Emerging Functions

“…A shuttle mechanism, as described above, likely operates in E-Syt as the 90Å-long SMP dimer is too short to span the distance between membranes ( Schauder et al, 2014 ). E-Syts were found to mediate the transport of glycerophospholipids in a bidirectional manner using in vitro assays ( Saheki et al, 2016 ; Yu et al, 2016 ; Bian et al, 2018 ; Bian and De Camilli, 2019 ). E-Syts might also participate in mechanisms altering lipid composition.…”