ABSTRACT
The ER forms contacts with other endomembrane systems to exchange materials (e.g., calcium and lipids) and also to modulate dynamic organelle processes, including fission, cargo sorting and movement. During autophagosome formation, dynamic contacts between the ER and the phagophore membrane are crucial for phagophore expansion and closure. Little is known about the mechanisms underlying the formation and disassembly of the ER contacts. We found that the ER-localized autophagy protein EPG-3/VMP1 plays an essential role in controlling ER-phagophore dissociation and also the disassembly of ER contacts with LDs, mitochondria and endolysosomes. VMP1 regulates the ER contact by activating the ER calcium channel ATP2A/SERCA (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting). CALM (calmodulin) acts as one of the downstream calcium effectors that controls the PIK3C3/VPS34 phosphatidylinositol (PtdIns) 3-kinase (PtdIns3K) activity to maintain these contacts. Our study provides insights into the molecular mechanisms which regulate ER contacts and generate autophagosomes.
KEYWORDS: ER, membrane contact, organelle, SERCA, VMP1
The ER forms dynamic contacts with other organelles, including lipid droplets (LDs), mitochondria, Golgi and endosomes. Formation of these contacts facilitates calcium transport and lipid exchange between the 2 compartments, and also plays a critical role in regulating organelle fission, fusion, maturation and movement. Different tether complexes have been identified to mediate the ER contacts with other membranes: DGAT2-SLC27A1/FATP1 for the ER-LD contact; VAPB-RMDN3/PTPIP51, MFN2-MFN1, ITPR/IP3R-HSPA9/GRP75-VDAC1 and BCAP31/BAP31-FIS1 for the ER-mitochondrion contact; VAPA-OSBP for the ER-Golgi contact; and VAPA-OSBPL1A/ORP1L and VAP-STARD3 for the ER-endosome contact. The interaction of proteins and phosphoinositides also functions in ER contact formation. For example, the PtdIns4P-binding activity of OSBP is essential for establishing the contact between the ER and Golgi. The mechanism controlling the disassembly of the ER contacts is largely unknown.
Autophagy is an evolutionarily conserved system for enclosing cytosolic contents within a double-membrane autophagosome and delivering them to lysosomes for degradation. Yeast genetic screens identified a set of autophagy-related (ATG) proteins that act at different steps of autophagosome formation. These proteins are conserved from yeast to mammals. In mammalian cells, these ATG proteins act sequentially to mediate autophagosome formation. Upon autophagy induction, the ULK1/Atg1-RB1CC1/FIP200 complex is recruited to autophagosome formation sites on the ER, which then recruit the PIK3C3/VPS34-BECN1-ATG14 PtdIns3K complex to generate phosphatidylinositol-3-phosphate (PtdIns3P)-enriched subdomains of the ER, known as omegasomes. The PtdIns3P effector WIPI2 is then recruited to trigger later steps of autophagosome generation. The formation of autophagosomes in higher eukaryotes is far more complicated, containing steps that are not present in yeast. One of the steps unique to autophagosome formation in more complex eukaryotes is the formation of extensive contacts between the ER and the phagophore to facilitate expansion of the latter. The molecular mechanism underlying the establishment, maintenance and disassembly of ER-phagophore contacts remains completely unknown.
Genetic screens in C. elegans identified a group of metazoan-specific autophagy genes, known as EPG genes, that are required for autophagy in more complex eukaryotes. Using a combination of imaging, biochemical and immunoEM analysis, we revealed that VMP1, the mammalian homolog of EPG-3, regulates the ER-phagophore contact during autophagosome formation. In VMP1 knockout (KO) cells, LC3-labeled autophagic structures stably colocalize with the ER-localized autophagic markers ZFYVE1/DFCP1 and RB1CC1, and also associate with the ER markers SEC61B/Sec61β and CANX, but are completely separable from LAMP1-labeled lysosomes. Levels of autophagy proteins in the purified microsome fractions from VMP1 KO cells are much higher than those from WT cells. ImmunoEM analysis revealed that double-membrane autophagic structures, labeled by gold particles recognizing LC3, remain associated with the ER in VMP1 KO cells. Thus, VMP1 modulates the disassembly of the ER-phagophore contact.
We further identified the tethering complex that mediates the ER-phagophore contact. In Wipi2 KO cells, LC3 puncta are separable from ZFYVE1/DFCP1-labeled omegasomes, suggesting that ER-phagophore contacts are disrupted by WIPI2 depletion. WIPI2 accumulates at the autophagosome formation sites in VMP1 KO cells. Simultaneous knockdown of Wipi2 suppresses the colocalization of LC3 puncta and ZFYCE1/DFCP1 in VMP1 KO cells. We demonstrated that WIPI2 interacts with the ULK1-RB1CC1 complex and the interactions are dramatically increased in VMP1 KO cells. WIPI2 is a PtdIns3P effector. Depletion of PtdIns3P by treatment with the PtdIns3K inhibitor wortmannin reduces the interaction of WIPI2 and RB1CC1 in VMP1 KO cells. Therefore, WIPI2 interacts with the ULK1-RB1CC1 complex on the ER and also with PtdIns3P on the ER and possibly on the phagophore to mediate ER-phagophore contacts.
To understand how VMP1 regulates ER-phagophore contacts, we performed coimmunoprecipitation assays and mass spectrometry analysis and found that VMP1 interacts with ATP2A2/SERCA2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2), an ER-localized calcium channel that transports calcium from the cytoplasm into the ER lumen. Inhibition of ATP2A/SERCA by its specific inhibitor thapsigargin (TG) also causes autophagy defects and persistent contacts between the ER and phagophores. The autophagy defect in TG-treated cells can be rescued by overexpression of an ATP2A/SERCA mutant with defective TG binding. The formation of an inhibitory complex between ATP2A/SERCA and its binding partners PLN and SLN is greatly enhanced by VMP1 KO and dramatically inhibited by overexpression of VMP1. Thus, VMP1 functions as an activator of ATP2A/SERCA. VMP1 directly competes with PLN and SLN to bind to ATP2A/SERCA, or stabilizes ATP2A/SERCA in its active form, which loses its capacity to bind with PLN and SLN. No ER stress or change in the cytosolic calcium level is elicited by depletion of VMP1 or treatment with TG (100 nM), suggesting that the autophagy defect in these cells results from local calcium perturbation.
In addition to the enhanced ER-phagophore contact, loss of VMP1 and TG treatment also increases the contact between the ER and other organelles, including LDs, mitochondria and endolysosomes. This indicates that local modulation of ATP2A/SERCA activity by VMP1 is a general mechanism for disassembly of ER contacts. CALM (calmodulin) appears to be one of the calcium effectors involved in contact regulation. Previous studies demonstrated that binding of PIK3C3/VPS34 with CALM and calcium is required for the PtdIns3K activity of PIK3C3/VPS34. CALM knockdown ameliorates the autophagy defect and partially suppresses the enhanced ER contacts in VMP1-depleted cells. Taken together, our data show that VMP1 acts as a general factor that modulates ER contacts with other organelles by activating ATP2A/SERCA activity.
In conclusion, our study reveals an essential step in autophagosome formation in more complex eukaryotes, namely the disassociation of contacts between the ER and phagophores. This process requires VMP1 to modulate the local calcium concentration via regulation of the ATP2A/SERCA activity. This mechanism also applies to the disassembly of ER contacts with other endomembrane systems.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.