The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation

Abstract

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

“…HeLa cells (Soni et al 2009), fibroblasts (Martin and Parton 2005), U937 cells (Wan et al 2007) and Huh7 cells (Fujimoto and Parton 2011). For HeLa cells the contact between LB and ER was quantified, showing that ER-lipid body and ER-endolysosome association is characteristic, deduced from single ultra-thin sections and confirmed with 3D reconstruction with TEM-tomography (Zhao et al 2017). Typical for LBs is the electron dense cortex, enhanced in contrast by fixation with ferrocyanide-reduced osmium/thiocarbohydrazide/osmium (= rOTO) also shown for HeLa cells by Zhao et al, (2017) and for human mast cells, however, fixed with 2% glutardialdehyde and 1% osmium tetroxide only (Dichlberger et al 2011).…”

TMEM 41B is a novel regulator of autophagy and lipid mobilization

“…An antibody directed against TMEM41B was unable to recognize the endogenous protein but confirmed a tubular ER pattern upon expression of untagged TMEM41B (Fig EV4A). The homology to VMP1 is interesting as this ER-localized transmembrane protein has been linked to the regulation of autophagy, LDs, and mitochondrial morphology [32,33]. This KI approach in HeLa cells confirmed that TMEM41B robustly co-localizes with the ER marker calnexin (Fig 5B).…”

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

“…In 2016, we described the association of VMP1 with diverse MCSs, thus suggesting possible roles in other cellular processes beyond autophagy . During the preparation of this manuscript, Zhao et al reported similar conclusions . Moreover, they found that VMP1 regulates calcium levels at MCSs by promoting the activity of the sarco‐endoplasmic reticulum calcium ATPAse.…”

“…The loss of perinuclear localization was confirmed by the using of the Golgi marker GM130, which colocalizes with SAC1 in the control cells and not as much in VMP1‐depleted cells (Figure B). Moreover, VAPA and the retromer subunit SNX2 extensively colocalized in these aberrant peripheral structures (Figure A), which may reflect the abnormally large ER‐endosome contact sites generated in the absence of VMP1 . The retromer complex mediates endosome‐to‐Golgi retrieval of various proteins, such as the mannose 6‐phosphate receptor .…”

“…It has been previously reported that ER-endosome and ER-mitochondria MCSs increase in the absence of VMP1, 18,35 which demonstrates that VMP1 is not essential for the formation of the contact itself, but may still be required for its functionality. Recently, Dong et al showed that the ER protein VAPA/B and the endosome protein SNX2 establish a platform for the LTP OSBP, which transports PtdIns4P generated by PI4K2A from endosomes to the ER.…”