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PMID:12065597
| Citation |
Mühlenhoff, U, Richhardt, N, Gerber, J and Lill, R (2002) Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP, NADH, and reduced iron. J. Biol. Chem. 277:29810-6 |
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| Abstract |
To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from Delta yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from Delta nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation. |
| Links |
PubMed Online version:10.1074/jbc.M204675200 |
| Keywords |
Adenosine Triphosphate/metabolism; Carrier Proteins/metabolism; Detergents; Iron/metabolism; Iron-Binding Proteins; Iron-Sulfur Proteins/metabolism; Membrane Potentials; Mitochondria/metabolism; NAD/metabolism; Oxidation-Reduction; Saccharomyces cerevisiae/metabolism |
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Significance
Annotations
| Gene product | Qualifier | GO ID | GO term name | Evidence Code | with/from | Aspect | Notes | Status |
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