ATM3 is required for the activity of cytosolic aconitase but not mitochondrial and plastid Fe-S enzymes. A, In-gel activities and protein blots of Arabidopsis aconitase isoforms in the wild type (WT) and aco mutants. Equal amounts of protein (80 μg) extracted from leaves were separated on nondenaturing gels containing 2% (w/v) starch/8% (w/v) PA in Tris-borate buffer and stained for aconitase activity (purple) as described in “Materials and Methods.” In addition, leaf protoplasts were disrupted and centrifuged at 12,000g to obtain pellet (P12) and supernatant (S12) fractions enriched in mitochondrial and cytosolic proteins, respectively. After separation on starch/PA gels, the fractions were blotted and labeled with aconitase antibodies (gray scale). B, In-gel aconitase activities (purple) and protein levels (gray scale) in wild-type, atm3, and atm1-1 leaves. For quantification, see Supplemental Figure S2. An equal volume (5 μL) of the native sample was separated on a denaturing gel, blotted, and labeled for actin as a loading control. C, RT-PCR of ACO1 and ACT8 in wild-type, atm3, and atm1-1 leaves. D and E, Activities of Fe-S enzymes in the cytosolic fraction (D) and purified mitochondria (E) of callus generated from roots of wild-type, atm3, and atm1-1 seedlings (n = 3). Based on fumarase activity measurements, approximately 40 milliunits of aconitase in the cytosolic fraction is estimated to be of mitochondrial origin (indicated by the asterisk). Error bars represent sd. F, NiR activity in leaf extracts of the wild type, atm3, and atm1-1 (n = 3). Error bars represent sd.