Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis

The budding yeast Saccharomyces cerevisiae contains two homologues of bacte rial IscA proteins, designated Isa1p and Isa2p, Bacterial IscA is a product of the ise (iron-sulfur cluster) operon and has been suggested to particip ate in Fe-S cluster formation or repair. To test the function of yeast Isa1 p and Isa2p, single or combinatorial disruptions were introduced in ISA1 an d ISA2. The resultant isa Delta mutants were viable but exhibited a depende ncy on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast gene s proposed to function in Fe-S cluster assembly, mitochondrial iron concent ration 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 o f Isa1p and Isa2p are essential for function and may be involved in iron bi nding. 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 seque nce that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p i n delivering iron to sites of the Fe-S cluster assembly is discussed.