Mitochondrial monothiol glutaredoxins that bind Fe-S Cluster are known to participate in Fe-S cluster assembly However, their precise role has not been Well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria The Delta grx5 mutant requires cysteine on minimal media. and does not grow oil non-fermentable carbon Source Such as glycerol We found that the Mutant IS low in the activity of Fe-S enzymes ill mitochondria as well as ill the cytoplasm Screening of multi-copy suppressor of growth defects of the mutant identified isaI(+) gene encoding a putative A-type Fe-S scaffold, in addition to mas5(+) and hsc1(+) genes encoding putative chaperones for Fe-S assembly process Examination Of Other scaffold and chaperone genes revealed that isa2(+). but not isu1(+) and ssc1(+), complemented the growth phenotype of Delta grx5 mutant as isa1(+) did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S pombe, and supports mitochondrial genome integrity as well as Fe-S assembly (C) 2010 Elsevier Inc All rights reserved.