The Molecular Mechanism Underlying Amino Acid Metabolism Regulates Other Metabolic Pathways In Mitochondria

Mitochondria are not only the powerhouse but also the metabolic center of the cell.Therefore,human health is inseparable from mitochondrial homeostasis and it's proper function.The study of this paper revolved around mitochondria,we designed a reporter to indicate mitochondria and endoplasmic reticulum contact sites(MERCs).Moreover,we investigated P5 CS,an enzyme catalyzes synthesis of pyrroliline-5-carboxylic acid(P5C)which is a key precursor for the synthesis of proline and ornithine,and the molecular mechanism underlying P5 CS regulates multiple metabolic pathways in mitochondria.MERCs are the site of calcium ion and phospholipid exchange between mitochondria and endoplasmic reticulum,and also the platform for the cross-talk of various signaling pathways between them.However,the acquaintance about the composition,distribution and dynamics of MERCs are very limited so far.The biggest difficulty in this field is lacking simple and practical markers could indicate MERCs.In the first part of this study,we constructed a novel reporter could indicate MERCs and analyzed the changes of MERCs under a series of stimulations with this reporter.The two parts of Split GFP: Split GFP1-10 and Split GFP11,can recombine into functional GFP and glow when they close enough.Based on this principle,we linked ER and mitochondrial targeting sequences to the sequences encoding Split GFP1-10 and Split GFP11 respectively.In the meantime,we added linkers with different lenghths between Split GFP11 and mitochondrial targeting sequence to fit the distance of MERCs.Overexpression the reporter in cells,Split GFP1-10 and Split GFP11 can recombine into functional GFP at MERCs and glow.We further observed and analyzed the distribution and dynamic of MERCs during mitotic cell cycle and under several stress conditions like starvation and ER stress.We found the dynamics of MERCs through living cellimages observation,which could disappear and rebuild within minutes.The states of mitochondria affected the abundace of MERCs.The abundance of MERCs were elevated when cells were under oxidative phospthorylation uncoupler: CCCP or mitochondrial ATPase inhibitor: oligomycin A treatment.While,there were no dramatic changes of MERCs when cells were under ER stress.Artificially stimulated apoptosis or autophagy could increase the level of cellular MERCs.Yet,fed oleic acid to drive cell lipid synthesis did not alter the distribution of MERCs.This study provided a fairly good tool for the research of MERCs.The second part of this paper focused on the enzymes involved in the synthesis of P5 C in mitochondria: pyrrolin-5-carboxylic acid synthase(P5CS).Mutations of genes encoding P5 CS cause multiple diseases in humans,but the pathogenesis is unclear.Here,we found that P5 CS localizes in mitochondria in rod-and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents.Some of the human disease related mutant forms of P5 CS also exhibit diffused distribution.We also found that multimerization(but not the catalytic activity)of P5 CS regulates its localization.P5 CS knockout cells have a reduced proliferation rate and are more sensitive to cellular stresses.Flies lacking P5 CS have reduced eclosion rates.Lipid droplets accumulate in the eyes of the newly eclosed P5 CS mutant flies,which degenerate with aging.Quasi-target metabolomics analysis showed that the loss of P5 CS in cells leads to abnormal purine metabolism and lipid metabolism.P5 CS knockout cells showed significant purinosomes and lipid droplet accumulations.The expression level of the fatty acid transporter,CPT1,and several ?-oxidation related genes were downregulated following P5 CS knockdown.Further investigations found that P5 CS knockout cells had defect of mitochondrial respiratory complex organization and that the respiration defects in P5 CS knockout cells likely contribute to the metabolicdefects in purine synthesis and lipid consumption.This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes,whose imbalance might contribute to P5CS-related disease conditions.