Fig 2: CIAO3 interactions are regulated by intracellular iron level in a time-dependent manner. A–C, Flp-In 293 stably expressing 3HA-3FLAG–tagged CIAO3 was induced with 1 μg/ml doxycycline for 24 h and treated with 100 μg/ml FAC or 100 μM DFO for 8 h before harvesting. Two biological replicates were performed for each experiment. A, affinity purified CIAO3 and associated proteins were characterized by shotgun proteomics and quantified by MS1 intensity-based label-free quantification. Abundance of co-eluted (B) CIA scaffold complex components (NUBP1 and NUBP2) and (C) CIA targeting complex components (MMS19, CIAO1, and CIAO2B) were monitored using a targeted proteomic assay. Log2 of calculated protein intensities (Log2Intensities) under FAC or DFO condition were plotted with mean ± SD. ∗ denotes p < 0.05. D, Flp-In 293 cells or Flp-In 293 expressing NUBP2 were induced with 1 μg/ml doxycycline for 24 h. Cells expressing 3HA-3FLAG-NUBP2 were treated with 100 μg/ml FAC, untreated or treated with 100 μM DFO for 8 h before harvesting. WCEs and anti-HA immunoprecipitates (IP: HA) were analyzed by immunoblotting with antibodies against FLAG, CIAO3, loading control α-tubulin, and FAC/DFO treatment control FTH1. E, Flp-In 293 background cells or cells stably expressing 3HA-3FLAG-CIAO3 were induced with 1 μg/ml doxycycline overnight, untreated, or treated with 100 μg/ml FAC or 100 μM DFO for hours indicated. WCEs and anti-HA immunoprecipitates (IP: HA) were blotted with antibodies against indicated proteins. CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; DFO, deferoxamine; FAC, ferric ammonium citrate; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.

Fig 3: Model for the regulated assembly of the CIA machinery in response to ROS, O2tension, intracellular iron levels, and Fe-S cluster assembly on CIAO3. CIA, cytosolic iron–sulfur cluster assembly; CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; ROS: reactive oxygen species.

Fig 4: Fe-S cluster incorporation into CIAO3 controls its interactions.A, schematic representation of Fe-S incorporation in wildtype CIAO3 and mutants. B–D, Flp-In 293 cells that stably express wildtype or mutant CIAO3 were induced with 1 μg/ml doxycycline for 24 h before harvesting. CIAO3 and associated proteins were affinity purified. Two biological replicates were analyzed. Protein abundances were monitored by a targeted proteomic assay containing known CIA components and a subset of prototypical CIA substrates after normalization to CIAO3 levels. Log2 transformed abundance of the co-eluted proteins (Log2Intensiteis) were plotted for the CIA scaffold complex (B), the CIA targeting complex (C), and selected CIA substrates (D). Mean ± SD was indicated. Protein levels of interactors co-eluted with mutant CIAO3 were compared to wildtype. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. E, Flp-In 293 control cells, cells expressing wildtype 3HA-3FLAG-ERCC2 and cells expressing mutant ERCC2 lacking the MMS19 binding region were induced with 1 μg/ml doxycycline for 24 h. Cell lysates were immunoprecipitated. WCEs and HA immunoprecipitates were blotted with indicated antibodies. CIA, cytosolic iron–sulfur cluster assembly; CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.

Fig 5: CIAO3 interactions are altered by changes in cellular redox state and requires functional mitochondrial Fe-S cluster biogenesis. A, Flp-In 293 cells expressing 3HA-3FLAG-CIAO3 were treated with tert-butyl hydroperoxide (tBHP) for 4 h to introduce reactive oxygen species. WCEs and anti-HA immunoprecipitates were blotted with antibodies against indicated proteins and loading control GAPDH. B, Flp-In 293 cells expressing 3HA-3FLAG-CIAO3 or control cells were induced with 1 μg/ml doxycycline overnight and cultured in 21% O2 or 1% O2 for 16 h before harvesting. WCEs and anti-HA immunoprecipitates were immunoblotted with antibodies against NUBP2, loading control α-tubulin, and hypoxia treatment control HIF1α. C, mitochondrial iron-sulfur cluster biogenesis was disrupted by using siRNA to silence ISCU1/2 in Flp-In 293 cells for 48 h while control cells were treated with nontarget siRNA. Doxycycline was added to induce expression of 3HA-3FLAG-CIAO3. WCEs and HA immunoprecipitates were blotted with indicated antibodies. D and E, quantification of (C) by densitometry. Protein abundance of coimmunoprecipitated proteins were normalized to the protein level of immunoprecipitated bait (3HA-3FLAG-CIAO3). Mean ± SD was plotted for n = 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01. CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.