PI(4,5)P2-Dependent and Ca2+-Regulated ER-PM Interactions Mediated by the Extended Synaptotagmins

Abstract

SUMMARY Most available information on ER-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca2+ entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here we show that the three Extended-Synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca2+ in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca2+ regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca2+ entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast), mediate the formation of ER-PM contacts sites which are functionally distinct from those mediated by STIM1 and Orai1.

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

“…The Arabidopsis SYT1 Localizes at the ER and the PM and Accumulates at ER-PM Contact Sites SYT1 contains a modular structure similar to ER-PM tether proteins such as E-Syts and tricalbins (Craxton, 2010;Yamazaki et al, 2010), proteins that are localized at specific ER-PM subdomains (Giordano et al, 2013;Helle et al, 2013;Prinz, 2014). Moreover, cell biological and biochemical analyses have reported SYT1 being at the ER, PM, and plasmodesmata (Schapire et al, 2008;Lewis and Lazarowitz, 2010;Yamazaki et al, 2010).…”

“…The SYT1 C2 Domains Are Targeted to the PM and Bind Negatively Charged Phosphatidylinositol Phosphates Similar to most tricalbins and E-Syts (Toulmay and Prinz, 2012;Giordano et al, 2013), SYT1 contains a predicted TM domain and phospholipid binding C2 domains through which it could connect the ER to the PM in trans. To assess the accuracy of these predictions and to distinguish between the two possible SYT1 orientations (TM anchored at the ER and C2 domains tethering the PM, or TM anchored at the PM and C2 domains tethering the ER), we first analyzed where isolated SYT1 C2 domains were targeted.…”

“…In mammals, the E-Syts C2 domains have been reported to bind phosphatidylinositol phosphates (PtdInsPs) in both a Ca 2+ -dependent and Ca 2+ -independent manner (Giordano et al, 2013). Once we determined that the SYT1 C2 domains were targeted to the PM, we aimed to identify phospholipid species that might be involved in the C2-PM tethering process.…”

“…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.…”

Inseparable tandem: evolution chooses ATP and Ca2+to control life, death and cellular signalling

“…Although operational SOCE is well characterized in the ciliate, Paramecium [40], Orai and Stim have not been detected (as yet), in contrast to informatics data obtained from choanoflagellates [78] that are placed at the basis of metazoans. However, several alternative modes of cell membranecortical store coupling are discussed, for instance by extended synaptotagmins with super-numerary C2 domains [133] which also occur in ciliates [38]. Altogether, SOCE appears to be an evolutionarily old mechanism.…”