Résumé
Autophagy is a critical intracellular degradation and recycling mechanism which supports plantacclimation and survival to a wide range of environmental stresses. During autophagy, the formation andmaturation of specialized membrane vesicles ensure cargo recognition, sequestration and trafficking to thelytic vacuole. This dynamic process relies on an orchestrated series of membrane remodeling events whichunderlying molecular mechanisms remain poorly understood.As essential components of membranes’ morphology, identity and activities, lipids have thepotential to functionally contribute to several steps of the autophagy pathway. To unravel these functions,we need to directly assess the nature and dynamics of the lipids composing the autophagy membranes.First, after induction of autophagy by nutrient starvation or pharmacological treatment, we immuno-isolatedautophagy structures in native conditions, using specific markers previously associated with either early orlate autophagy structures. Quality control analyses combining western-blot, immunolabeling and electrontomography showed a high level of purity of the resulting membrane fractions. Second, by combining GCMSand LC-MS analyses, fatty acids methyl esters, sterols, glycerolipids and sphingolipids of the purifiedmembranes were analyzed. These studies established the lipid cartography of autophagic compartments,showing its robustness in regard to autophagy inducing conditions and stage of the pathway, andhighlighting its molecular specificities compared to other intracellular compartments. Third, proteomicsanalyses of the purified fractions identified singularities between the proteomes of early vs late autophagycompartments while cross-analyses of these proteomes showed a specific enrichment in proteins involvedin autophagy, vesicle and membrane trafficking as well as lipid metabolism. Transient expression of asubset of these proteins confirmed their co-localization with autophagy markers supporting their potentialfunction in the autophagy process.Together, our data contribute to the understanding of the machinery of autophagy and the originsof the autophagy membranes. Additionally, this work provides specific lipids and lipid-related proteins whichfunctional characterization is in progress to unravel their contribution in membrane remodeling andautophagy.