Abstract
Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.
Highlights
Mitochondria are the center of numerous metabolic functions of primary importance to the cell
Model System—The protein content of mitochondria isolated from sibling cybrid cell lines, homoplasmic either for wild-type or for the Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mutation A8344G in the tRNALys gene, has been compared
Proteomic maps of mammalian mitochondria become progressively presented in the case of human placenta [34] and rat liver [35]
Summary
Human osteosarcoma-derived cybrid cell lines R2-1A and R1C3 were a kind gift of A. Cell lines were grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 100 g/ml bromodeoxyuridine at 37 °C in the presence of 5% CO2 and 85% humidity. R2-1A cells were seeded at a density of 2 ϫ 105 cells/10 ml of medium/10-cm diameter Petri dish, and R1C3 cells were seeded at a density of 5 ϫ 105 cells/10 ml/10 cm diameter. Cells were passed (not more than 4 or 5 times), and 50 –100 dishes, containing ϳ4 ϫ 106 cells each (confluent culture), were harvested for mitochondria isolation. Stability of the cell lines was verified in regard to the tRNALys and tRNALeu(UUR) gene sequences, which were found to be unchanged over the period of experimentation
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