Characterization of iron-sulfur protein assembly in isolated mitochondria -: A requirement for ATP, NADH, and reduced iron

被引:107
作者
Mühlenhoff, U [1 ]
Richhardt, N [1 ]
Gerber, J [1 ]
Lill, R [1 ]
机构
[1] Univ Marburg, Inst Zytobiol & Zytopathol, D-35033 Marburg, Germany
关键词
D O I
10.1074/jbc.M204675200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Deltayfh1 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 Deltanfu1 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 an vitro reconstitution of the entire pathway of Fe/S protein maturation.
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页码:29810 / 29816
页数:7
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