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PUX10 associates with CDC48A and regulates the dislocation of ubiquitinated oleosins from seed lipid droplets
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International audience. In oleaginous seeds, storage lipids are packed in organelles called lipid droplets (LDs) and are degraded post-germinatively to provide carbon and energy for seedling growth. The major LD-associated proteins, called oleosins, form a protective barrier that shields stored lipids from lipases, thus preventing lipolysis. The degradation of this barrier begins just prior lipid degradation in germinating seeds. Oleosin ubiquitination was recently shown to control oleosin degradation in Arabidopsis seedlings1. Ubiquitination of oleosins is surprisingly complex. Three distinct and exclusive motifs can be attached to the major oleosins OLE1 and OLE2 and may channel modified oleosins towards specific degradation pathways according to ubiquitination type. Once ubiquitinated, oleosins are extracted from the LD coat before proteolysis. The molecular mechanism of oleosin extraction is yet unknown. To gain insight to the proteins involved in oleosin extraction, we analyzed proteomic data obtained from LDs isolated in Arabidopsis germinated seeds. We identified a putative ubiquitin-binding protein and an AAA ATPases protein called PUX10 and CDC48A, respectively. To explore their role in LD dynamics, we undertook functional analyses using T-DNA mutants. Because CDC48A-deficient mutants were reported as lethal2, we focused our study on PUX10 mutants. We showed that PUX10 mutation blocks the degradation of all the three types of ubiquitinated oleosins, and prevents the dislocation of ubiquitinated oleosins from LDs, which precedes the degradation. By confocal microscopy and organelle fractionation, we confirmed that PUX10 is an integral LD protein and we identified its membrane-anchoring domain. By microscopy, Y2H, Y3H and BiFC, we demonstrated that PUX10 interacts with ubiquitin and CDC48A through its UBA and UBX domains, respectively. Overall, our data revealed that PUX10 is an adaptor bridging ubiquitinated oleosins and CDC48A, thus allowing the ubiquitin-selective « segregase » activity of CDC48A to separate ubiquitinated proteins from LDs.
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