Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.The seed is a complex, specific structure that allows a quiescent plant embryo to cope with unfavorable germinating conditions and also permits dissemination of the species. To achieve these functions, seeds accumulate reserve compounds that will ensure the survival of the embryo and fuel the growth of the plantlet upon germination. Accumulation of lipids occurs in many eukaryotic cells and is a rather common means of storing carbon and energy. Lipid droplets (LDs) can be found in all eukaryotes, such as yeast (Saccharomyces cerevisiae;
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.
In contrast with melanocytes, melanomas display constitutive expression of HLA-DR (HLA-DR+). This abnormal expression has been associated with tumour progression and metastatic dissemination. We have previously reported that this deregulation of HLA-D genes is due to the abnormal constitutive expression of the lymphocyte-specific isoform of class II transactivator (B-CIITA), in addition to its fibroblast form (F-CIITA), which is usually expressed in major histocompatibility complex (MHC) class II-negative interferon-gamma-induced cell types, such as melanocytes. In this study, we investigated the abnormal expression of B-CIITA in a panel of melanoma cell lines displaying differential HLA-DR expression profiles, and analysed whether such a molecular event can participate in tumour progression. Our results showed that the abnormal expression of B-CIITA did not have any particular effect, in comparison with F-CIITA, on the classical activity of CIITA HLA-D gene regulation. As CIITA has also been shown to regulate genes other than HLA-D, we evaluated the modulation of those encoding cyclin D1, YARS (tyrosyl-tRNA synthetase) and TRIP1 (transforming growth factor (TGF)-beta receptor-interacting protein), proteins involved in cell cycle/apoptosis balance, angiogenesis and resistance to TGF-beta, respectively. In contrast with other cell types, neither B-CIITA nor F-CIITA was able to modulate these genes in melanoma cell lines. Thus, the activity of CIITA, whether lymphocyte-specific or fibroblast-specific, is restricted to HLA-D gene expression in these tumours. Accordingly, our data suggest that CIITA is not involved per se in tumour progression; rather, it is the MHC class II molecules themselves, through tumour antigen presentation and the induction of tumour antigen-specific CD4 lymphocyte anergy, that may participate in immune escape and melanoma progression.
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