Cells lacking Nfu1 exhibit defects in [4Fe-4S] cluster enzymes in mitochondria. (A) Respiratory growth defects revealed by yeast drop-test. Cells harboring empty vectors (EV) or high-copy plasmids expressing designated genes were pre-cultured in liquid synthetic complete (SC) glucose media lacking uracil. Serially diluted cells (10-fold) were spotted on SC media plates at 30°C. Grx5 is a monothiol glutaredoxin involved in mitochondrial Fe-S biogenesis. Isa1, Isa2 and Iba57 are subunits of the ISA scaffold complex required for [4Fe-4S] cluster synthesis. Yap1 is a transcription factor that induces expression of anti-oxidant genes. Glu is 2% glucose and Ace is 2% acetate. (B) The relative activity of aconitase, SDH, cytochrome bc1, cytochrome c oxidase (CcO), and malate dehydrogenase (MDH) were measured in isolated mitochondria from cells cultured in SC media with 2% raffinose. Data are shown as mean ± SE (n = 3) (CcO, n = 4). (C) Steady-state protein levels measured by SDS-PAGE followed by immunoblotting in isolated mitochondria. Anti-LA antibody is an antibody specific to lipoic acid (LA) that is conjugated to proteins. PDH is pyruvate dehydrogenase and KDH is α-ketoglutarate dehydrogenase. Sdh2 is the Fe-S cluster subunit of SDH. Aco1 is mitochondrial aconitase. Por1 is a mitochondrial loading control. (D) Restoration of LA moieties on PDH and KDH shown by SDS-PAGE followed by immunoblotting in isolated mitochondria from nfu1∆ cells over-expressing ISA1 and ISA2. (E) Enzymatic activity of SDH in mitochondria isolated from nfu1∆ cells over-expressing ISA1 and ISA2. Data are shown as mean ± SE (n = 3). (F) Strep-tag affinity purification of Nfu1-Strep revealed the Nfu1 interaction with Isa1 and Isa2. Mitochondria were solubilized with 0.1% n-dodecyl maltoside (DDM). Clarified lysates were incubated with Strep-Tactin superflow beads for 16 hr. After washing, proteins were eluted with 2.5 mM desthiobiotin, and then analyzed by immunoblotting. (G) Strep-tag affinity purification of Nfu1-Strep in the presence of ectopically expressed Aco2-HA. Nfu1m-Strep is the G/T>H mutant described in Figure 4. (H) Strep-tag affinity purification of Nfu1-Strep in the presence of ectopically expressed Lys4-HA. Lys4 and Aco2 are both nuclear DNA-encoded mitochondrial proteins that require a [4Fe-4S] cluster for each function in the lysine biosynthetic pathway in yeast. Nfu1m-Strep is the G/T>H mutant described in Figure 4.