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

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

doi:10.1080/15548627.2020.1834207

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

Autophagy. 2021 Sep;17(9):2528-2548. doi: 10.1080/15548627.2020.1834207. Epub 2020 Oct 23.

Authors

Valeria Manganelli¹, Paola Matarrese², Manuela Antonioli³, Lucrezia Gambardella², Tiziana Vescovo³, Christine Gretzmeier⁴, Agostina Longo¹, Antonella Capozzi¹, Serena Recalchi¹, Gloria Riitano¹, Roberta Misasi¹, Joern Dengjel^{4

5}, Walter Malorni⁶, Gian Maria Fimia^{3

7}, Maurizio Sorice¹, Tina Garofalo¹

Affiliations

¹ Department of Experimental Medicine, Sapienza University, Rome, Italy.
² Oncology Unit, Center for Gender-Specific Medicine, Istituto Superiore di Sanità, Rome, Italy.
³ Department of Epidemiology, Preclinical Research and Advanced Diagnostics, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy.
⁴ Department of Dermatology, Faculty of Medicine, Medical Center-University of Freiburg, Freiburg, Germany.
⁵ Department of Biology, University of Fribourg, Suisse, Germany.
⁶ School of Pharmacy, University of Tor Vergata, Rome, Italy.
⁷ Department of Molecular Medicine, Sapienza University, Rome, Italy.

Abstract

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.

Keywords: AMBRA1; ERLIN1; autophagy; lipid rafts; mitochondria associated membranes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Autophagosomes* / metabolism
Autophagy* / genetics
Lipids
Mitochondria / metabolism
Mitochondrial Membranes / metabolism

Substances

Lipids

Grants and funding

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].