Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP, NADH, and reduced iron.
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.
Mühlenhoff
Ulrich
U
Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch Strasse 5, 35033 Marburg, Germany.
Richhardt
Nadine
N
Gerber
Jana
J
Lill
Roland
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D003902:Detergents; D007501:Iron / Q000378:metabolism*; D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D000096923:Frataxin
D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D003902:Detergents; D007501:Iron; D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins; D009243:NAD; D000096923:Frataxin