RESUMO
Mitochondria share attributes of vesicular transport with their bacterial ancestors given their ability to form mitochondrial-derived vesicles (MDVs). MDVs are involved in mitochondrial quality control and their formation is enhanced with stress and may, therefore, play a potential role in mitochondrial-cellular communication. However, MDV proteomic cargo has remained mostly undefined. In this study, we strategically used an in vitro MDV budding/reconstitution assay on cardiac mitochondria, followed by graded oxidative stress, to identify and characterize the MDV proteome. Our results confirmed previously identified cardiac MDV markers, while also revealing a complete map of the MDV proteome, paving the way to a better understanding of the role of MDVs. The oxidative stress vulnerability of proteins directed the cargo loading of MDVs, which was enhanced by antimycin A (Ant-A). Among OXPHOS complexes, complexes III and V were found to be Ant-A-sensitive. Proteins from metabolic pathways such as the TCA cycle and fatty acid metabolism, along with Fe-S cluster, antioxidant response proteins, and autophagy were also found to be Ant-A sensitive. Intriguingly, proteins containing hyper-reactive cysteine residues, metabolic redox switches, including professional redox enzymes and those that mediate iron metabolism, were found to be components of MDV cargo with Ant-A sensitivity. Last, we revealed a possible contribution of MDVs to the formation of extracellular vesicles, which may indicate mitochondrial stress. In conclusion, our study provides an MDV proteomics signature that delineates MDV cargo selectivity and hints at the potential for MDVs and their novel protein cargo to serve as vital biomarkers during mitochondrial stress and related pathologies.
Assuntos
Mitocôndrias Cardíacas/fisiologia , Estresse Oxidativo , Vesículas Transportadoras/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mioblastos , Proteômica , RatosRESUMO
Iron (Fe) is an essential nutrient for virtually all organisms, where it functions in critical electron transfer processes, like those involved in respiration. Photosynthetic organisms have special requirements for Fe due to its importance in photosynthesis. While the importance of Fe for mitochondria- and chloroplast-localized processes is clear, our understanding of the molecular mechanisms that underlie the trafficking of Fe to these compartments is not complete. Here, we describe the Arabidopsis mitochondrial iron transporters, MIT1 and MIT2, that belong to the mitochondrial carrier family (MCF) of transport proteins. MIT1 and MIT2 display considerable homology with known mitochondrial Fe transporters of other organisms. Expression of MIT1 or MIT2 rescues the phenotype of the yeast mrs3mrs4 mutant, which is defective in mitochondrial iron transport. Although the Arabidopsis mit1 and mit2 single mutants do not show any significant visible phenotypes, the double mutant mit1mit2 displays embryo lethality. Analysis of a mit1 -- /mit2 + - line revealed that MIT1 and MIT2 are essential for iron acquisition by mitochondria and proper mitochondrial function. In addition, loss of MIT function results in mislocalization of Fe, which in turn causes upregulation of the root high affinity Fe uptake pathway. Thus, MIT1 and MIT2 are required for the maintenance of both mitochondrial and whole plant Fe homeostasis, which, in turn, is important for the proper growth and development of the plant.