GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells.

Marina Besprozvannaya,Hao Li,Kenneth S Ginburg,Donald M Bers,Jodi Nunnari,Johan Auwerx,Eamonn Dickson

doi:10.7554/elife.31019

Abstract

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

Highlights

The endoplasmic reticulum (ER) in eukaryotic cells is a vast and distributed intracellular network of membranous tubes and sheets with essential roles in ion and lipid homeostasis
Both GRAMD1a-eGFP and GRAMD2a-eGFP were observed in focal structures at the periphery of Cos7 cells, suggesting that, similar to previously characterized yeast Ltc3/4 (Lam4/ Ysp2) proteins, they localize to Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (Figure 1C and D)
GRAMD2a is a simple GRAM domain tether that is targeted to the PM via interaction of its GRAM PH-like domain with PIP lipid determinants – a mechanism shared by a majority of ER-PM tethers (Figure 8) (Chung et al, 2015; Heo et al, 2006; Raychaudhuri et al, 2006; Stefan et al, 2011)

Summary

Introduction

The endoplasmic reticulum (ER) in eukaryotic cells is a vast and distributed intracellular network of membranous tubes and sheets with essential roles in ion and lipid homeostasis. It exerts many of its functions through intimate membrane contact sites (MCSs) with other organelles (Prinz, 2014). MCSs between the ER and the plasma membrane (ER-PM) are a highly conserved feature of eukaryotic cells and have emerged as key regulators of intracellular Ca2+ dynamics (Chang and Liou, 2016; Dickson et al, 2016a; Eisenberg-Bord et al, 2016; Henne et al, 2015; Saheki and De Camilli, 2017a; Stefan et al, 2013).

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