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Genome-wide Responses to Mitochondrial Dysfunction

Genome-wide Responses to Mitochondrial Dysfunction. overview. Transcriptional changes due to disruption of mitochondrial function. Two different yeast strains: S. cerevisiae lacking mtDNA ()….(petite). Respiratory deficient, obligatory fermentative metabolism.

jesse-lawson
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Genome-wide Responses to Mitochondrial Dysfunction

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  1. Genome-wide Responses to Mitochondrial Dysfunction

  2. overview Transcriptional changes due to disruption of mitochondrial function • Two different yeast strains: • S. cerevisiae lacking mtDNA ()….(petite). • Respiratory deficient, obligatory fermentative metabolism. • Wild-type S. cerevisiae (+). • Respiratory competent, partially oxidative metabolism. Compared genome-wide expression with microarrays. Compared with previous microarray experiment looking at genome-wide changes in expression in yeast cells undergoing diauxic shift.

  3. yeast carbon metabolism Aerobic metabolism: (oxidative metabolism) Glycolysis TCA cycle oxidative phosphorylation Anaerobic metabolism: (fermentation) Glucose  Acetyl CoA  Ethanol Diauxic shift: Metabolic change as fermentable carbon source is used up from… Glucose Fermentative (Glycolysis  Ethanol) to… Oxidative Metabolism (Ethanol  TCA cycle)

  4. differential gene expression • Clusters D and E: • intermediary metabolism • & small molecule • transport pathways • in  and diauxic shift Reflect metabolic changes to compensate for loss of respiration in petite cells. • Clusters A and B: • Oxidative phosphorylation • Apparatus • in petite  No attempt by cells to upregulate oxidative metabolism • in diauxic shift Change to oxidative metabolism in shift • Cluster C: • Cytoplasmic ribosomal • Proteins • in diauxic shift Shows slowing of growth rate upon change to oxidative metabolism

  5. metabolic remodelling Metabolic pathways were altered in respiratory deficient cells Intermediates of TCA cycle needed for synthesis of amino acids and nucleotides Oxaloacetate (OAA) is not regenerated in petite () cells’ TCA cycle, so must be replenished another way. Metabolic pathways altered:  OAA and Acetyl-CoA supply  Acetyl-CoA hydrolase  in enzymes involved in flux and conversion of metabolites made by fatty acid oxidation to TCA and glyoxylate cycle intermediates.  in nutrient and metabolite transporters.  in enzymes for reoxidation of NADH  petites reconfigure metabolism by recruiting peroxisomal activities, small molecule transport systems and lipid, sugar and amino acid turnover to get more OAA and Acetyl-CoA.

  6. effect of mitochondrial inhibitors Different transcriptional responses due to different mitochondrial inhibitors. Antimycin: effects are similar to petite cells. CCCP: different response. Oligomycin: different response. Petite expression profile shows effects of loss of electron transport rather than loss of mitochondrial ATP synthesis.

  7. peroxisome proliferation In  petites, peroxisome biogenesis is induced. peroxisome targeted GFP used to trace peroxisomes in  cells. Antimycin also induced peroxisome biogenesis.

  8. RTG genes RTG genes important in retrograde regulation. RTG1, 2 & 3 upregulate peroxisomal citrate synthase in  petits. RTG genes increase expression of TCA cycle enzymes in  petits. Array profiling of  cells with mutations in each RTG gene. Cluster A: Genes induced in  petits whose induction is dependent on RTG signalling pathway. Cluster B: Genes requiring RTG genes for expression in  petits. Cluster C: Genes showing enhanced expression in  petits without RTG genes. Cluster D: Genes showing enhanced expression in  petits independent of RTG genes, indicating novel retrograde regulation pathways.

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