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. 2018 Mar 9;293(21):8297–8311. doi: 10.1074/jbc.RA118.001885

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Figure 3. — Widespread loss of Fe-S and lipoate-dependent proteins in cells expressing dominant–negative ISCU mutants. A, cells expressing ISCU2^D71A or ISCU2^C69S for either 24 or 36 h showed decreased protein levels of Fe-S enzymes in mitochondria (FECH), cytosol (PPAT), and nucleus (NTHL1) and a robust decrease in mitochondrial and cytosolic aconitase activities. B, analysis of complex II activity by in-gel activity assay revealed decreased SDH enzyme activity in cells expressing ISCU2^D71A or ISCU2^C69S. Immunoblots demonstrated decreased SDHB protein levels, but SDHA protein levels were unchanged in cells expressing ISCU2^D71A or ISCU2^C69S. Immunoblotting of TOM20 served as the loading control for mitochondrial proteins. C, in-gel assay of NADH-tetrazolium reductase activity showed decreased complex I activity in cells expressing ISCU2^D71A or ISCU2^C69S for 24 and 36 h. Immunoblotting showed decreased abundance of the complex I core subunit, NDUFS1, but no change in levels of cysteine desulfurase NFS1. Immunoblots revealed decreased protein levels of LIAS and decreased lipoylation of the subunits of pyruvate dehydrogenase (DLST) and α-ketoglutarate dehydrogenase (DLAT) (D) and GCSH (E). F, further immunoblotting showed that total protein levels of these subunits were not decreased.