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

Abstract

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca 2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipidbinding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt-mediated ER-PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Sytdependent contacts were by far the predominant contacts, ER-PM distance (19-22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca 2+ shortened the ER-PM distance at E-Syt1-dependent contacts sites. E-Syt-mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca 2+ channel Orai1 as well as store operated Ca 2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt-dependent ER-PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.T he endoplasmic reticulum (ER) consists of a complex network of tubules and cisternae that extends throughout the cell and forms close appositions ("contact sites") with other membranous organelles and with the plasma membrane (PM) (1, 2). The best characterized function of ER-PM contacts is in Ca 2+ homeostasis, as ER-PM contacts mediate the excitationcontraction coupling in muscle and store-operated Ca 2+ entry (SOCE) in all metazoan cells. In SOCE, upon depletion of Ca 2+ in the ER, the ER protein stromal interaction molecule 1 (STIM1) oligomerizes, binds, and activates Orai1 Ca 2+ channels at the PM to drive influx of extracellular Ca 2+ , thereby allowing homeostatic regulation of ER Ca 2+ levels (3, 4). However, growing evidence suggests that ER-PM contacts also play more general roles, including signaling (5, 6) and the regulation of both lipid metabolism and transport between bilayers (1, 7-17).We recently have shown that the three mammalian extended synaptotagmins (E-Syts), homologs of the yeast tricalbins (18,19), act as ER-PM tethers (20). 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 loc...

TMEM110 regulates the maintenance and remodeling of mammalian ER–plasma membrane junctions competent for STIM–ORAI signaling

“…2C and see below) (20). The TIRF-layer ER signal does have the limitation of including contributions both from true cortical ER in close contact with the plasma membrane (5,11,12,24,25) and from adjacent ER that is within the TIRF layer. However, its unmatched advantage, which came into sharp focus as the experiments progressed, is the ability to monitor near-plasma membrane ER over the entire footprint of a cell, with high spatial resolution and over the time course of a physiological response.…”

“…2C) (22,30). This comingling of calcium signaling and lipid transfer reflects a close functional linkage: Lipid transfer supplies the phosphatidylinositol needed for PIP2 resynthesis during sustained calcium signaling, and plasma membrane lipid microdomains are integral to feedback regulation of the ORAI channel by calcium and SARAF (22,31,32); conversely, local calcium signals recruit E-Syt1 and thereby either expand ER-plasma membrane junctions or strengthen junctional contacts (11,12,22,30).…”

“…Even subtle structural changes in existing junctions can shape calcium signaling both by altering the local landscape for calcium diffusion, and hence the local calcium concentration profile, and by setting constraints on the positioning of calcium-handling proteins such as SERCA and PMCA (6). Recent mechanistic studies have highlighted the dynamic changes attributable to E-Syt proteins and their partners (11,12,22,30). Here we establish that TMEM110 and STIM2, proteins dedicated primarily to calcium signaling, also participate in rapid structural rearrangements of junctional contacts (Figs.…”

“…For example, the formation and maintenance of ER-plasma membrane junctions depends on tricalbins and other proteins in yeast cells (8)(9)(10) and in mammalian cells on the extended synaptotagmins (E-Syts), a family of three tricalbin homologs, and other unidentified proteins (11,12). The insights from yeast extend to the mechanisms of cellular lipid metabolism and lipid transfer between the apposed bilayers that have been conserved from yeast cells to human cells (13)(14)(15)(16)(17)(18)(19).…”

Extended synaptotagmins (E-Syts): Architecture and dynamics of membrane contact sites revealed

“…Using a clever combination of mutant and wild-type protein overexpression, Fernández-Busnadiego et al (3) report that contacts mediated by E-Syts are distinguished by a prominent electron-dense layer between (and parallel to) the membranes. Comparison of various E-Syts and E-Syt mutants indicates that the layer depends on the presence of the C2A, C2B, and SMP domains.…”