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Abstract
The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1and ISA2. The resultant isaΔ mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced. An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding. As predicted, Isa1p is targeted to the mitochondrial matrix. However, Isa2p is present within the intermembrane space of the mitochondria. Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed.
ACKNOWLEDGMENTS
We are indebted to K. Yu and J. Boeke for generating the isa1Δ strain and for noting the lysine auxotrophy associated with this mutant. We also thank J. Boeke for critical review of the manuscript, L. Vickery and R. Jensen for helpful discussions and for the cytochrome b 2 and Mas2 antibodies, and A. Dancis for the fet3::URA3 plasmid.
This work was supported by the JHU NIEHS center and by NIH grant GM 50016 to V.C.C. L.T.J. was supported by NIEHS training grant ES 07141.
ADDENDUM IN PROOF
In a recent study by Lill and colleagues (A. Kaut, H. Lang, K. Diekert, G. Kispal, and R. Lill, J. Biol. Chem., in press) S. cerevisiae Isa1p was shown to be important for mitochondrial iron metabolism.