Structure of a lipid-bound extended synaptotagmin indicates a role in lipid transfer

Abstract

Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions.

Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

“…This model cannot be excluded, because we observed very few focal contacts with ER-PM distances less than 10 nm. However, the larger ER-PM distances observed at the great majority of E-Syt-dependent contacts are more consistent with the shuttle model (23), in which lipids first are extracted by the SMP domain at one membrane and then are delivered to the other membrane by a translocation of the SMP domain kept in place by C2 domain-dependent interactions. Cytosolic Ca 2+ may regulate such exchanges, because all E-Syts exhibit Ca 2+ -dependent properties: Although the ER-PM tethering function is strongly regulated by Ca 2+ only in the case of E-Syt1 (because the properties of its C2C domain; see ref.…”

“…This shortening from C2E-to C2C-mediated PM binding could be consistent with the magnitude of the change in the ER-PM distance that we quantified. The property of E-Syts to harbor phospholipids in their SMP domains (23), the localization of several SMP domain-containing proteins at membrane contact sites (19), and the similarity of SMP modules to modules whose known function is to exchange lipids (21, 23) strongly suggest that E-Syts have a role in lipid exchange between membranes. Furthermore, the peculiar elongated structure of the 9-nm-long SMP domain obligate dimer, with a deep hydrophobic groove that spans the entire domain from tip to tip and nests the acyl groups of phospholipids, had suggested the possibility that lipid exchange may be achieved by direct flow of lipids from one bilayer to the other via the SMP domain bridge [the tunnel model of lipid exchange (23)].…”

“…E-Syts are ER-anchored proteins (via an N-terminal hairpin) containing a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain followed by five (E-Syt1) or three (E-Syt2/3) C2 domains. SMP domains are present in proteins that localize at various organelle contact sites, where they have been implicated in lipid transfer between bilayers (19,(21)(22)(23). As shown by a recent structural and mass spectrometry study of E-Syt2, the SMP domain dimerizes to form a ∼90-Å-long cylinder traversed by a longitudinally oriented channel lined with hydrophobic residues that harbor glycerophospholipids (23).…”

“…SMP domains are present in proteins that localize at various organelle contact sites, where they have been implicated in lipid transfer between bilayers (19,(21)(22)(23). As shown by a recent structural and mass spectrometry study of E-Syt2, the SMP domain dimerizes to form a ∼90-Å-long cylinder traversed by a longitudinally oriented channel lined with hydrophobic residues that harbor glycerophospholipids (23). C2 domains typically are membranebinding modules whose interaction with the bilayer often is potentiated by elevations in cytosolic Ca 2+ and/or facilitated by the presence of acidic phospholipids.…”

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

“…To achieve their tethering function, most yeast tricalbins and mammalian E-Syts establish ER-PM contact sites in durable or transient states using a single aminoterminal ER anchor (Manford et al, 2012), the SMP domain that localizes them to specific ER-PM subdomains (Toulmay and Prinz, 2012), and a variable number of phospholipid binding domains (C2 domains) that connect the proteins to the PM in trans via electrostatic interactions with negatively charged phospholipids (Toulmay and Prinz, 2011;Manford et al, 2012;Giordano et al, 2013;Schauder et al, 2014). Our results suggest that the SYT1 localization at the nexus between the ER and the PM is restricted by its TM and SMP domains, and its docking to the PM mainly occurs through nonspecific electrostatic interaction with negatively charged PtdInsPs (Fig.…”