PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis.

Postgerminative mobilization of neutral lipids stored in seed lipid droplets (LDs) is preceded by the degradation of oleosins, the major structural LD proteins that stabilize LDs in dry seeds. We previously showed that Arabidopsis thaliana oleosins are marked for degradation by ubiquitination and are extracted from LDs before proteolysis. However, the mechanisms underlying the dislocation of these LD-anchored proteins from the LD monolayer are yet unknown. Here, we report that PUX10, a member of the plant UBX-domain containing (PUX) protein family, is an integral LD protein that associates with a subpopulation of LDs during seed germination. In pux10 mutant seedlings, PUX10 deficiency impaired the degradation of ubiquitinated oleosins and prevented the extraction of ubiquitinated oleosins from LDs. We also showed that PUX10 interacts with ubiquitin and CDC48A, the AAA ATPase Cell Division Cycle 48, through its UBA and UBX domains, respectively. Collectively, these results strongly suggest that PUX10 is an adaptor recruiting CDC48A to ubiquitinated oleosins, thus facilitating the dislocation of oleosins from LDs by the segregase activity of CDC48A. We propose that PUX10 and CDC48A are core components of a LD-associated degradation machinery, which we named the LD-associated degradation system. Importantly, PUX10 is also the first determinant of a LD subpopulation described in plants, suggesting functional differentiation of LDs in Arabidopsis seedlings.

Reserve lipids and plant autophagy.

Autophagy is a universal mechanism that facilitates the degradation of unwanted cytoplasmic components in eukaryotic cells. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals. We consider the storage compartments that form the sources of lipids in plants, and the roles that autophagy plays in the synthesis of triacylglycerols and in the formation and maintenance of lipid droplets. We also consider the relationship between lipids and the biogenesis of autophagosomes, and the role of autophagy in the degradation of lipids in plants.

Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis.

In oleaginous seeds, lipids--stored in organelles called oil bodies (OBs)--are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub(2)) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.

Barley γ3-hordein: glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat.

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.

E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.

It has now been clearly shown that lipid droplets (LDs) play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+) ATPase (V-ATPase). Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

A recombinant ω-gliadin-like D-type glutenin and an α-gliadin from wheat (Triticum aestivum): two immunoglobulin E binding proteins, useful for the diagnosis of wheat-dependent allergies.

Among the wheat prolamins, D-type glutenins display a highly repetitive sequence similar to ω-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An ω-gliadin-like D-type glutenin, an α-gliadin, and an ω5-gliadin-like D-type glutenin were obtained as recombinant proteins and compared using synchrotron radiation circular dichroism. This technique evidenced the strong thermostability of the ω5-gliadin-like protein. The IgE reactivity of recombinant proteins was evaluated using 45 sera from wheat-allergic patients. The sera from patients diagnosed with cutaneous hypersensitivity to hydrolyzed wheat proteins often reacted with the ω-gliadin-like D-type glutenin and α-gliadin, whereas the IgE reaction was less frequent after dietary sensitization. So, these two proteins could be useful to diagnose these diseases. The sera from patients with exercise-induced anaphylaxis recognized the ω5-gliadin-like protein as a positive control and, less frequently, the other proteins tested. Only some sera from patients with baker's asthma reacted with the proteins tested.