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Proc Natl Acad Sci U S A. 2015 Apr 21;112(16):E2004-13. doi: 10.1073/pnas.1503191112. Epub 2015 Mar 18.

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

Author information

1
Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510.
2
Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510 pietro.decamilli@yale.edu.

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-lipid-binding 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-Syt-dependent 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.

KEYWORDS:

E-Syt; TULIP; cryo-electron microscopy; lipid transfer; phosphoinositides

PMID:
25787254
PMCID:
PMC4413308
DOI:
10.1073/pnas.1503191112
[Indexed for MEDLINE]
Free PMC Article

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