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Raft-like lipid microdomains drive autophagy initiation via AMBRA1-ERLIN1 molecular association within MAMs

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posted on 13.10.2021, 18:08 by Valeria Manganelli, Paola Matarrese, Manuela Antonioli, Lucrezia Gambardella, Tiziana Vescovo, Christine Gretzmeier, Agostina Longo, Antonella Capozzi, Serena Recalchi, Gloria Riitano, Roberta Misasi, Joern Dengjel, Walter Malorni, Gian Maria Fimia, Maurizio Sorice, Tina Garofalo

Mitochondria-associated membranes (MAMs) are essential communication subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. We previously demonstrated that, upon macroautophagy/autophagy induction, AMBRA1 is recruited to the BECN1 complex and relocalizes to MAMs, where it regulates autophagy by interacting with raft-like components. ERLIN1 is an endoplasmic reticulum lipid raft protein of the prohibitin family. However, little is known about its association with the MAM interface and its involvement in autophagic initiation. In this study, we investigated ERLIN1 association with MAM raft-like microdomains and its interaction with AMBRA1 in the regulation of the autophagic process. We show that ERLIN1 interacts with AMBRA1 at MAM raft-like microdomains, which represents an essential condition for autophagosome formation upon nutrient starvation, as demonstrated by knocking down ERLIN1 gene expression. Moreover, this interaction depends on the “integrity” of key molecules, such as ganglioside GD3 and MFN2. Indeed, knocking down ST8SIA1/GD3-synthase or MFN2 expression impairs AMBRA1-ERLIN1 interaction at the MAM level and hinders autophagy. In conclusion, AMBRA1-ERLIN1 interaction within MAM raft-like microdomains appears to be pivotal in promoting the formation of autophagosomes.

Abbreviations: ACSL4/ACS4: acyl-CoA synthetase long chain family member 4; ACTB/β-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATG14: autophagy related 14; BECN1: beclin 1; CANX: calnexin; Cy5: cyanine 5; ECL: enhanced chemiluminescence; ER: endoplasmic reticulum; ERLIN1/KE04: ER lipid raft associated 1; FB1: fumonisin B1; FE: FRET efficiency; FRET: Förster/fluorescence resonance energy transfer; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GD3: aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)ceramide; HBSS: Hanks’ balanced salt solution; HRP: horseradish peroxidase; LMNB1: lamin B1; mAb: monoclonal antibody; MAMs: mitochondria-associated membranes; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MYC/cMyc: proto-oncogene, bHLH transcription factor; P4HB: prolyl 4-hydroxylase subunit beta; pAb: polyclonal antibody; PE: phycoerythrin; SCAP/SREBP: SREBF chaperone; SD: standard deviation; ST8SIA1: ST8 alpha-N-acetyl-neuraminide alpha-2,8 sialyltransferase 1; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUBB/beta-tubulin: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VDAC1/porin: voltage dependent anion channel 1.


This work was supported by grants from the Italian Association for Cancer Research [18526 to PM, 17404 to GMF]; PRIN project 2015 [20152CB22L to GMF], PRIN project 2017 [2017FS5SHL to MS] and PRIN project 2017 [2017SNRXH3 to RM]; Fondazione Umberto Veronesi [individual-fellowship 2017 to MA] and the Nando and Elsa Peretti Foundation [3603 to PM].