Reconstitution and biochemical studies of extended synaptotagmin-mediated lipid transport

Extended synaptotagmins (E-Syts) are a family of lipid transfer proteins (LTPs) located at the endoplasmic reticulum (ER)-plasma membrane (PM) contact sites in eukaryotic cells. They possess a conserved synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and two to five C2 domains. While the membrane tethering function of E-Syts has been well studied in diverse species, recent studies revealed that the mammalian E-Syt1 and its yeast homolog tricalbin 3 (Tcb3) could transport lipids between the opposed membrane. Mechanical studies suggested SYT1 transfers lipids fundamentally through the SMP domain, but the lipid transport requires the regulation of C2 domain-mediated membrane tethering. In addition, both E-Syt1 and Tcb3 are Ca²⁺-modulated LTPs, which sense and interact with Ca²⁺ through the C2 domains. This chapter describes the in vitro reconstitution and biochemical assays for studying the functions and mechanisms of E-Syts, by expressing and purifying recombinant proteins, preparing reconstitution systems, and developing assays for membrane tethering and lipid transport.