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Development and validation of a new method for autophagosomes purification and characterization from plant cells

Lemoine, Denis
(2022)

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Lemoine_36361700_2022.pdf
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Lemoine_36361700_2022_Annexe1.pdf
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Details

Supervisors
Henri Batoko ; Michel Ghislain
Faculty
Faculté des bioingénieurs
Degree label
Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée
Abstract
Macroautophagy, hereafter autophagy, is a highly conserved and ubiquitous process in eukaryotic cells that degrade cytoplasmic components in response to nutrient starvation and cellular stresses. Cytoplasmic components to be degraded are engulfed by a de novo formed double-membrane vesicle called autophagosome. Autophagosome formation is a sequential and tightly regulated process. Autophagy is required for plant adaptation in various biotic or abiotic stress conditions. However, the molecular mechanisms of plant autophagy signaling are not well understood, leaving a big challenge to control this important pathway to enhance plant growth and development. Understanding these pathways will require full characterization of the proteins involved and the possible integration hubs, leading to a great interest to develop an efficient method for identification and characterization of autophagosome content and regulatory factors involved in their trafficking and fusion with the lytic vacuole. This goal has not been achieved yet for any eukaryotic cell type due in part to a lack of robust and comprehensive autophagosome purification method. Indeed, deciphering the proteome of autophagosome will require a reasonable amount of autophagosomes at the highest possible purity level. We show herein that an original approach of autophagosome purification can yield a plant autophagosomes-enriched preparation suitable for proteomic analysis and identification of defined autophagosome set proteome. With the validated design and approach coupled to our purification method and mass spectrometry, we are in position to envisage the proteome characterization of autophagosomes generated as a response to different stress conditions and/or cell type from plant.