title: Events upstream of mitochondrial protein import limit the oxidative capacity of fibroblasts in multiple mitochondrial disease.
abstract: To investigate whether protein import is defective in mitochondrial disease, we compared the rate of import and the expression of protein import machinery components in skin fibroblasts from control subjects and a patient with multiple mitochondrial disease (MMD). The patient exhibited a 35% decrease in cytochrome c oxidase activity and a 59% decrease in cellular oxygen consumption compared to control. Western blot analyses revealed that patient levels of MDH, mtHSP70, HSP60, and Tom20 protein were 57%, 20%, 75% and 100% of control cells, respectively. MDH and Tom20 mRNA levels were not different from control levels, whereas mtHSP70 mRNA were 50% greater than control. Radiolabeled MDH was imported into mitochondria with equal efficiency between patient (44% of total synthesized) and control (43%) cells, although the total MDH synthesized in patient cells was reduced by about 40%. The unaffected levels of mRNA and post-translational import into mitochondria, combined with reduced protein levels of MDH, mtHSP70, and HSP60 suggest a translational defect in this patient with MMD. This was verified by the 50% reduction in overall cellular protein synthesis in the patient compared to control. Further, the similar import rates between patient and control cells suggest an important role for Tom20, but a lesser role for mtHSP70 in regulating protein import into mitochondria.
authors.affiliation: Department of Kinesiology, Toronto, ON, Canada M3J 1P3.
authors.forename: Arne A
authors.initials: AA
authors.lastname: Rungi
authors.forename: Andy
authors.initials: A
authors.lastname: Primeau
authors.forename: Lorraine
authors.initials: L
authors.lastname: Nunes Christie
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Gordon
authors.forename: Brian H
authors.initials: BH
authors.lastname: Robinson
authors.forename: David A
authors.initials: DA
authors.lastname: Hood
mesh_terms: D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000032:analysis / Q000378:metabolism; D051546:Cyclooxygenase 2; D004272:DNA, Mitochondrial / Q000032:analysis; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D026901:Membrane Transport Proteins; D028361:Mitochondrial Diseases / Q000201:enzymology / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface; D013462:Sulfur Radioisotopes; D015534:Trans-Activators; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D029867:Xenopus Proteins
chemical_list: D018834:Chaperonin 60; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D013462:Sulfur Radioisotopes; C499631:TFAM protein, human; C084008:TOMM20 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C097078:XL-MTTFA protein, Xenopus; D029867:Xenopus Proteins; C422562:mitochondrial transcription factor A; D008291:Malate Dehydrogenase; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases