Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used sucrose gradient centrifugation and Blue-Native PAGE to separate mitoribosomes of Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates.
Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.
Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.
Project description:Diplonema papillatum represents a group of highly diverse and abundant marine protists with still unknown lifestyle and ecological functions. Based on alterations of the transcriptomic, proteomic and metabolomic profiles obtained from cells grown under different conditions we designed a metabolic map of its cellular bioenergetic pathways. Comparative analysis in the nutrient-rich and -poor media and in the absence and presence of oxygen revealed a capacity for major metabolic reprograming. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation and synthesis of fatty acid. While gluconeogenesis uniquely dominates over glycolysis, TCA cycle represents a combination of standard and unusual enzymes. The presence of typical anaerobic enzymes such as pyruvate:NADP+ oxidoreductase, fumarate reductase, opine dehydrogenase, enoyl-coenzyme A reductase, and lactate dehydrogenase reflects the ability to survive in low-oxygen environments. The metabolism quickly reacts to restricted carbon source, revealing unusual flexibility of diplonemids, also reflected in cell morphology and motility, which is in good correlation with their extreme ecological valence.