Figure 1.

ISCU mutants disrupt cell growth and mitochondrial morphology. A–C, model of the Fe-S cluster ligating residues in the ISCU scaffold and predicted positions of the altered residues in the mutant proteins. D, immunoblots of total protein lysates showing expression of ISCU and the accessory Fe-S assembly factors cysteine desulfurase NFS1, the structural protein ISD11, and frataxin. *, predicted uncleaved mitochondrial precursor proteins; **, predicted protein degradation fragments. E, immunoprecipitation of recombinant ISCU protein using anti-Myc beads showed that NFS1 and ISD11, but not frataxin, were bound in a stable complex. F, real-time cell proliferation analysis revealed no change in cell growth following induction of ISCU2^WT protein expression, whereas induction of ISCU2^D71A or ISCU2^C69S expression resulted in diminished cell proliferation. Arrows, time of addition of either vehicle (water) or 1 μg/ml doxycycline. Cell growth was monitored using an Incucyte FLR instrument (see “Experimental procedures”; n = 5, mean ± S.D.). G–J, EM demonstrated significant mitochondrial swelling and distorted cristae in cells induced to express ISCU2^D71A or ISCU2^C69S for 24 h.