Fig 1: CIA components and substrates form higher order complexes. A, HEK293 cells were treated with 100 µg/ml ammonium ferric citrate (FAC) or 100 µM deferoxamine (DFO) for 8 h to create an iron-sufficient or -deficient environment, respectively. Whole cell extracts (WCEs) were resolved by SDS-PAGE and blotted with antibodies against known components of the cytosolic iron–sulfur cluster assembly (CIA) pathway, CIA substrates, loading control a-tubulin, FAC treatment control FBXL5, and DFO treatment control IREB2. B, Flp-In T-Rex 293 cell line (Flp-In 293) engineered to stably express NUBP2 was induced overnight with 1 µg/ml doxycycline. Affinity purified NUBP2 and associated proteins were identified by bottom-up proteomics. Data-dependent acquisition of two technical replicates was performed. Spectra count (SpC) for selected proteins combined from the two technical replicates was shown. C, parallel reaction monitoring was conducted on HEK293 whole cell lysate with indicated amounts of protein using a targeted proteomics assay that monitors the abundance of known CIA factors (ABCB7, GLRX3, BOLA2, CIAPIN1, NUBP1, NUBP2, CIAO3, CIAO1, CIAO2B, CIAO2A, MMS19) and prototypical substrates (CDKAL1, DNA2, ERCC2, POLD1, and ABCE1). Two technical replicates were acquired. Intensities were normalized for each precursor to the highest intensity in a replicate. D, Flp-In 293 cells that stably express NUBP2 were induced with 1 µg/ml doxycycline. Affinity purified NUBP2 and associated proteins were identified by acquisition of a targeted proteomic assay containing CIA scaffold complex components (NUBP1 and NUBP2), CIAO3, CIA targeting complex components (MMS19, CIAO1, and CIAO2B) and prototypical substrates (CDKAL1, DNA2, ERCC2, POLD1, and ABCE1). Two biological replicates were performed. Acquired spectra were searched with MaxQuant. Each edge represents a peptide identified. Solid edges connect bait protein to known interactors while dashed edges connect to novel interactors discovered in our study. Edge widths correspond to the posterior error probability of each peptide. E, Flp-In 293 background cells or cells expressing NUBP2 were either directly harvested or after treatment with 1% formaldehyde. Anti-HA immunoprecipitation were performed. WCEs and anti-HA immunoprecipitates were blotted for CIAO3, CIA targeting complex components, and the CIA substrate CDKAL1. CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2.