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

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/ß-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.