Events upstream of mitochondrial protein import limit the oxidative capacity of fibroblasts in multiple mitochondrial disease.
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.
Rungi
Arne A
AA
Department of Kinesiology, Toronto, ON, Canada M3J 1P3.
Primeau
Andy
A
Nunes Christie
Lorraine
L
Gordon
Joseph W
JW
Robinson
Brian H
BH
Hood
David A
DA
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
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