Unveiling the molecular identity of plant autophagic compartments: A proteo-lipidomic study in Arabidopsis thaliana

Autophagy is an intracellular catabolic and recycling process critical for plant stress tolerance. Upon autophagy induction, a unique and very specialized membrane named phagophore nucleates, shapes, expands and fuses to yield a double membrane vesicle sequestering and trafficking cargo for degradation. To better understand how this intense series of membrane remodeling events is orchestrated and identify the underlying actors, we aimed at establishing the molecular footprint of the phagophore. In this study, we first report on a method to purify autophagic compartments in Arabidopsis, by combining cell fractionation and immuno-isolation in native conditions. Second, proteomic analyses of ATG-isolated membranes allowed us to establish a list of candidate proteins involved in autophagy, membrane remodeling, vesicular trafficking and lipid metabolism and colocalizing with autophagic compartments after transient expression in Nicotiana benthamiana. Third, lipidomic analyses revealed that ATG-isolated membranes are mostly composed of glycerophospholipids-with a minor contribution of sterols and sphingolipids-, including a large proportion of phosphatidylcholine and phosphatidylglycerol. Together, our study unravels the singular composition of the plant phagophore and provides key protein and lipid candidates to explore how lipid and membrane dynamics instruct the autophagy pathway.