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PUX10 associates with CDC48A and regulates the extraction of ubiquitinated oleosins from seed lipid droplets
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International audience. Seed lipid droplets (LDs) of many dicotyledonous plants, including Arabidopsis thaliana, are a good model to investigate the mechanisms controlling LD dynamics. Indeed, LD degradation occurs rapidly and massively in germinated seeds, to provide carbon and energy for seedling growth. Post-germinative mobilization of storage lipids is preceded by the degradation of oleosins, the major structural LD proteins which stabilize LDs in dry seeds. We previously showed that oleosins are marked for degradation by ubiquitination, and extracted from LDs before proteolysis. The mechanisms underlying the dislocation of these LD-anchored proteins from the phospholipid monolayer are yet unknown. Here, we report that a novel LD-associated protein, called PUX10, control this process. We phenotypically characterized and functionally complemented two mutants of PUX10 in Arabidopsis. Using biochemical approaches, we showed that PUX10 deficiency delays the degradation of oleosins and ubiquitinated oleosins, and prevents the extraction of ubiquitinated oleosins from the LD surface. We also identified several functional domains by expressing truncated versions of PUX10 in tobacco leaves or in yeast. In addition to a membrane-anchoring domain, PUX10 possesses two functional UBA and UBX domains that mediate interactions with ubiquitin and the CDC48A AAA-ATPase, respectively. We propose that PUX10 is a LD-anchored scaffolding protein recruiting CDC48A to ubiquitinated oleosins, thus promoting the dislocation of oleosins from LDs by the segregase activity of CDC48A. This mechanism is reminiscent of the ER-associated degradation (ERAD) translocation process but localizes to LDs; we therefore propose to name it LD-associated degradation (LDAD). Importantly, our comprehensive analysis of PUX10 localization using Arabidopsis transgenic lines expressing PUX10-GFP under native promoter revealed that PUX10 is progressively expressed and sequentially targeted to a specific subpopulation of LDs in germinated seeds. PUX10 is therefore the first determinant of a LD subpopulation identified in plants, suggesting a functional differentiation of LDs.
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