Résumé
Autophagy is a quality control mechanism highly conserved in eukaryotes. This pathway allows the degradation of cell components including proteins or organelles and the recycling of the resulting macromolecules which supports cellular homeostasis and plant survival during environmental stresses. This dynamic process relies on several membrane remodeling events starting with the de novo assembly of an initial membrane structure, the phagophore, which then expands by addition of lipids, surrounding its cargo, until its closure into a double-membrane vesicle termed autophagosome. Upon completion, the autophagosome fuses with the vacuolar membrane delivering its cargo into the lytic lumen of the vacuole for degradation. Despite their critical function in autophagy and plant physiology, the molecular mechanisms supporting these membrane remodeling events remain poorly understood. Autophagy is governed by a group of proteins named AuTophaGy related proteins (ATG), which functions are now well described in A. thaliana. However, little is known about the contribution of membrane lipids to autophagy. In order to unravel the functions of lipids in autophagosome formation, we first aimed at characterizing the nature and levels of the lipids and proteins that compose their membranes. After induction of autophagy by nutrient starvation or pharmacological treatment, we immunoisolated autophagy structures, in native conditions, using two different specific protein markers. Quality control using western-blot and proteomics showed a high level of purity of the resulting membrane fractions and unraveled the proteome associated with autophagy structures. Further, cross-analyses between the proteome of autophagic structures isolated with ATG8a and that isolated with ATG18a highlighted a specific enrichment in proteins involved in autophagy, vesicle and membrane trafficking as well as lipid metabolism. Transient expression of a subset of these proteins in N. benthamiana confirmed their co-localization with autophagy structures supporting their potential function in the autophagy process. Finally, lipid analyses of the purified membranes were initiated using GC-MS and LC-MS. Notably, the profile of fatty acids methyl esters, sterols and glycerolipids was determined unraveling the lipid mapping of the autophagic structures and highlighting the molecular differences with other previously characterized membrane compartments. Together, these data contribute to the foundation of broad knowledge on the machinery of autophagy and provides specific lipid and protein targets for structural and functional studies of the molecular mechanisms governing autophagosome formation.