Abstract
Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biological Assay
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Cytochrome Reductases / genetics
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Cytochrome Reductases / metabolism*
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Disulfides / chemistry
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Disulfides / metabolism
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Fibroblasts / cytology
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Fibroblasts / metabolism*
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Gene Expression Regulation
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Glutathione / metabolism*
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HEK293 Cells
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Humans
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Kinetics
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Membrane Potential, Mitochondrial / physiology
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Mice
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Mitochondria / metabolism*
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Mitochondrial Membrane Transport Proteins / genetics
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Mitochondrial Membrane Transport Proteins / metabolism*
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Mitochondrial Membranes / metabolism*
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Mitochondrial Precursor Protein Import Complex Proteins
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Mitochondrial Proteins / genetics
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Mitochondrial Proteins / metabolism
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Oxidation-Reduction
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Oxidoreductases Acting on Sulfur Group Donors
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Protein Folding
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Protein Stability
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Protein Transport
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Proteolysis
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Signal Transduction
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Sulfur Radioisotopes
Substances
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CHCHD4 protein, human
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COX19 protein, human
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Disulfides
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Mitochondrial Membrane Transport Proteins
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Mitochondrial Precursor Protein Import Complex Proteins
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Mitochondrial Proteins
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Sulfur Radioisotopes
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Cytochrome Reductases
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GFER protein, human
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Oxidoreductases Acting on Sulfur Group Donors
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Glutathione