Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery
2022; Elsevier BV; Volume: 298; Issue: 7 Linguagem: Inglês
10.1016/j.jbc.2022.102094
ISSN1083-351X
AutoresXiaorui Fan, William D. Barshop, Ajay A. Vashisht, Vijaya Pandey, Stephanie Leal, Shima Rayatpisheh, Yasaman Jami‐Alahmadi, Jihui Sha, James A. Wohlschlegel,
Tópico(s)RNA modifications and cancer
ResumoThe cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization. The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization. Iron–sulfur (Fe-S) clusters are ubiquitous cofactors utilized by all realms of life, among which [2Fe-2S] and [4Fe-4S] clusters are the most commonly found in biological systems (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar). These cofactors play a role in maintaining protein stability, as well as regulating subcellular localization and enzymatic activity (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, 3Ben-Shimon L. Paul V.D. David-Kadoch G. Volpe M. Stümpfig M. Bill E. et al.Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.J. Cell Sci. 2018; 131: jcs211433Crossref PubMed Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature's modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). Being redox sensitive, these clusters also serve as redox centers to facilitate electron transfer. The redox states of Fe-S clusters change in response to environmental stimuli, which provides an additional layer of regulation of protein function (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature's modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). In eukaryotic organisms, the biogenesis of Fe-S clusters is highly compartmentalized with distinct branches of the biogenesis pathway responsible for the maturation of mitochondrial and extramitochondrial Fe-S proteins (5Maio N. Rouault T.A. Outlining the complex pathway of mammalian Fe-S cluster biogenesis.Trends Biochem. Sci. 2020; 45: 411-426Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Lill R. Srinivasan V. Mühlenhoff U. The role of mitochondria in cytosolic-nuclear iron-sulfur protein biogenesis and in cellular iron regulation.Curr. Opin. Microbiol. 2014; 22: 111-119Crossref PubMed Scopus (104) Google Scholar). The maturation of extramitochondrial Fe-S proteins is facilitated specifically by the cytosolic Fe-S cluster biogenesis pathway (CIA). The CIA pathway is associated with a plethora of cellular processes, including cell proliferation, DNA damage repair, nonsense-mediated decay, apoptosis, and microtubule-based processes such as ciliogenesis (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. 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Lysandrou C. et al.The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis.Cell Mol. Life Sci. 2014; 71: 517-538Crossref PubMed Scopus (21) Google Scholar, 11Stehling O. Netz D.J. Niggemeyer B. Rösser R. Eisenstein R.S. Puccio H. et al.Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.Mol. Cell Biol. 2008; 28: 5517-5528Crossref PubMed Scopus (94) Google Scholar). Deregulation of CIA components and substrates has also been linked to numerous human diseases (5Maio N. Rouault T.A. Outlining the complex pathway of mammalian Fe-S cluster biogenesis.Trends Biochem. Sci. 2020; 45: 411-426Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Sheftel A. Stehling O. Lill R. Iron-sulfur proteins in health and disease.Trends Endocrinol. Metab. 2010; 21: 302-314Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13Fuss J.O. Tsai C.L. Ishida J.P. Tainer J.A. Emerging critical roles of Fe-S clusters in DNA replication and repair.Biochim. Biophys. Acta. 2015; 1853: 1253-1271Crossref PubMed Scopus (177) Google Scholar). The maturation of cytosolic Fe-S proteins is a multistep process that is tightly regulated. In human cells, bioavailable iron is delivered for [2Fe-2S] cluster biogenesis by poly(rC)-binding protein 1 (PCBP1) to the chaperone consisting of the BolA-like protein 2 (BOLA2) and glutaredoxin-3 (GLRX3) (14Patel S.J. Frey A.G. Palenchar D.J. Achar S. Bullough K.Z. Vashisht A. et al.A PCBP1–BolA2 chaperone complex delivers iron for cytosolic [2Fe–2S] cluster assembly.Nat. Chem. Biol. 2019; 15: 872-881Crossref PubMed Scopus (59) Google Scholar). [4Fe-4S] clusters are first assembled on the CIA scaffold complex composed of nucleotide-binding protein 1 (NUBP1) and nucleotide-binding protein 2 (NUBP2) (11Stehling O. Netz D.J. Niggemeyer B. Rösser R. Eisenstein R.S. Puccio H. et al.Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.Mol. Cell Biol. 2008; 28: 5517-5528Crossref PubMed Scopus (94) Google Scholar, 15Netz D.J. Pierik A.J. Stümpfig M. Mühlenhoff U. Lill R. The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol.Nat. Chem. Biol. 2007; 3: 278-286Crossref PubMed Scopus (152) Google Scholar). This step requires an unknown sulfur containing compound that is produced by the mitochondrial Fe-S cluster biogenesis (ISC) machinery and transported to the cytosol through the mitochondrial inner membrane protein ABCB7. This transiently bound [4Fe-4S] cluster is then transferred to the cluster carrier protein cytosolic iron–sulfur assembly component 3 (CIAO3) and eventually incorporated into apoprotein substrates through the activity of the CIA targeting complex composed of MMS19 nucleotide excision repair protein homolog (MMS19); probable cytosolic iron-sulfur protein assembly protein CIAO1 (CIAO1); cytosolic iron-sulfur assembly component 2B (CIAO2B) (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, 16Gari K. León Ortiz A.M. Borel V. Flynn H. Skehel J.M. Boulton S.J. MMS19 links cytoplasmic iron-sulfur cluster assembly to DNA metabolism.Science. 2012; 337: 243-245Crossref PubMed Scopus (183) Google Scholar). Although the crosstalk has been extensively documented in numerous organisms between Fe-S cluster biogenesis and the cellular environment such as intracellular iron and oxygen levels, evidence supporting this idea is only just beginning to emerge in humans (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, 4Beinert H. Holm R.H. Munck E. Iron-sulfur clusters: nature's modular, multipurpose structures.Science. 1997; 277: 653-659Crossref PubMed Scopus (1547) Google Scholar). The availability of bioavailable iron was recently shown to regulate cytosolic [2Fe-2S] cluster biogenesis by controlling the association of BOLA2 and GLRX3 (17Frey A.G. Palenchar D.J. Wildemann J.D. Philpott C.C. A glutaredoxin-BolA complex serves as an iron-sulfur cluster chaperone for the cytosolic cluster assembly machinery.J. Biol. Chem. 2016; 291: 22344-22356Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Additionally, the maturation of specific extramitochondrial Fe-S proteins involved in DNA repair and iron homeostasis are regulated by iron and oxygen availability (18Vashisht A.A. Yu C.C. Sharma T. Ro K. Wohlschlegel J.A. The association of the xeroderma pigmentosum group D DNA helicase (XPD) with transcription factor IIH is regulated by the cytosolic iron-sulfur cluster assembly pathway.J. Biol. Chem. 2015; 290: 14218-14225Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 19Meyron-Holtz E.G. Ghosh M.C. Rouault T.A. Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo.Science. 2004; 306: 2087-2090Crossref PubMed Scopus (207) Google Scholar). Despite these advances, however, the mechanisms underlying much of this regulation are still unknown. In this work, we developed a targeted proteomics assay that monitor proteins in the CIA pathway. Using this assay, we were able to detect the association of the CIA targeting complex (MMS19, CIAO1, and CIAO2B) and CIA substrates (DNA2, POLD1, CDKAL1, and ERCC2) with the CIA scaffold complex component NUBP2. We also find that the interaction of CIAO3 with the CIA scaffold complex is regulated by acute changes in cellular environment, including changes in the labile iron pool, exposure to reactive oxygen species (ROS), and changes in oxygen tension. The interaction of CIAO3 with the CIA targeting complex, although minimally affected by these acute environmental changes, is dependent on Fe-S cluster binding by CIAO3. CIAO3 mutants that are defective in Fe-S cluster binding display impaired association with the rest of the CIA machinery. Together, these data suggest the formation of CIA metabolon composed of the CIA scaffold complex, CIAO3, the CIA targeting complex and CIA substrates. The metabolon assembly is dynamic and regulated by environmental cues, possibly through altering Fe-S clusters in CIAO3. In order to investigate how the CIA pathway responds to changes in cellular environment, we began by comparing the endogenous protein levels of major CIA components in cells exposed to iron supplementation or chelation, mimicking an iron sufficient or deficient environment. Cells were treated with ferric ammonium citrate (FAC) or the iron chelator deferoxamine mesylate (DFO) for 8 h, and whole cell lysates were probed with antibodies against CIA components as well as two known substrates. Protein levels for F-box/LRR-repeat protein 5 (FBXL5), an E3 ligase that accumulates when sufficient iron is present, and iron-responsive element-binding protein 2 (IREB2/IRP2), an FBXL5 substrate that is stabilized by iron depletion, served as treatment controls for FAC and DFO, respectively (20Vashisht A.A. Zumbrennen K.B. Huang X. Powers D.N. Durazo A. Sun D. et al.Control of iron homeostasis by an iron-regulated ubiquitin ligase.Science. 2009; 326: 718-721Crossref PubMed Scopus (320) Google Scholar). We did not observe significant changes in the steady-state levels of either CIA components or CIA substrates in this time frame (Fig. 1A). Although defects in Fe-S cluster incorporation have been previously shown to cause destabilization of a number of Fe-S proteins, these effects are typically observed either as a result of chronic ablation of the Fe-S cluster assembly machinery or in mutant proteins defective in cluster incorporation (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, 21Golinelli M.P. Chmiel N.H. David S.S. Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY.Biochemistry. 1999; 38: 6997-7007Crossref PubMed Scopus (56) Google Scholar). Since we did not observe any immediate effect of changes in intracellular iron levels on the stability of CIA factors and substrates, we next examined interactions between CIA components under basal conditions. Multiple independent studies have shown that components of the CIA targeting complex are detected in higher order complexes ranging in molecular weight from 400 to 1000 kDa (16Gari K. León Ortiz A.M. Borel V. Flynn H. Skehel J.M. Boulton S.J. MMS19 links cytoplasmic iron-sulfur cluster assembly to DNA metabolism.Science. 2012; 337: 243-245Crossref PubMed Scopus (183) Google Scholar, 22Seki M. Takeda Y. Iwai K. Tanaka K. IOP1 protein is an external component of the human cytosolic iron-sulfur cluster assembly (CIA) machinery and functions in the MMS19 protein-dependent CIA pathway.J. Biol. Chem. 2013; 288: 16680-16689Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Kim K.S. Maio N. Singh A. Rouault T.A. Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAO1-mediated transfer to recipients.Hum. Mol. Genet. 2018; 27: 837-852Crossref PubMed Scopus (32) Google Scholar). Components of the CIA targeting complex interact with CIAO3, which in turn interacts with the CIA scaffold complex, indicating that these CIA components may be organized into higher order complexes. To examine this possibility, we performed affinity purification of NUBP2, a component of the CIA scaffold complex, and characterized proteins associated with NUBP2 using an unbiased shotgun proteomic approach. In addition to NUBP1 and CIAO3 which are known NUBP2 interactors, we also detected two components of the CIA targeting complex (CIAO1 and MMS19) as well as CIA substrates (CDKAL1 and ELP3) in the NUBP2 immunoprecipitates (Fig. 1B and Table S1). CIAO2B, another component of the targeting complex, was not identified in this analysis, although this could be due to poor sampling of low abundance peptides. To address this potential issue, we developed a targeted proteomics assay (tier 3) that utilizes parallel reaction monitoring (PRM) to assess the presence and abundance of a panel of proteins relevant to the Fe-S cluster assembly pathways (24Carr S.A. Abbatiello S.E. Ackermann B.L. Borchers C. Domon B. Deutsch E.W. et al.Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.Mol. Cell Proteomics. 2014; 13: 907-917Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). We first tested this assay on a serially diluted peptide standard prepared from HEK293 whole cell extracts and were able to detect the presence and estimate the relative abundance of known CIA factors (ABCB7, BOLA2, GLRX3, CIAPIN1, NUBP1, NUBP2, CIAO3, MMS19, CIAO1, CIAO2B, and CIAO2A) and several prototypical CIA substrates (ABCE1, CDKAL1, ERCC2, and POLD1) (Fig. 1C). This targeted approach, which provides better sensitivity and quantitation than unbiased proteomics assays, was then applied to NUBP2 immunoprecipitates to specifically monitor diagnostic peptides derived from components of the CIA scaffold complex (NUBP1 and NUBP2), CIAO3, components of the CIA targeting complex (MMS19, CIAO1, and CIAO2B), and CIA substrates (CDKAL1, DNA2, ERCC2, ABCE1, and POLD1). Diagnostic peptides utilized in this analysis are listed in Table S2. As expected, NUBP1 and CIAO3 were identified as interacting proteins (Fig. 1D and Table S3). All components of the CIA targeting complex (MMS19, CIAO1, and CIAO2B) were also found associated with NUBP2. In addition, we observed that CIA substrates CDKAL1, DNA2, ERCC2, and POLD1 copurified with NUBP2 (Fig. 1D). These observations together suggest that the CIA scaffold complex, CIAO3, the CIA targeting complex, and CIA substrates potentially assemble into a higher order protein assembly that facilitates Fe-S cluster transfer into substrates. The assembly is likely dynamic, given that formaldehyde crosslinking enhanced the association between the CIA scaffold complex and the CIA targeting complex (Fig. 1E). To determine the influence of intracellular iron availability on the assembly of CIA complexes, we utilized affinity purification of CIAO3 followed by tandem mass spectrometry to compare the CIAO3 interactome between iron-replete and iron-depleted conditions. These proteomics studies were done using both standard unbiased protein identification followed by label-free quantitation as well as our targeted proteomics assay that specifically measures the abundance of a panel of key Fe-S machinery proteins and substrates. The unbiased proteomics analysis demonstrated that interactions between CIAO3 and multiple proteins depend on the availability of labile iron, including both components of the CIA scaffold complex (NUBP1 and NUBP2) (Fig. 2A and Table S4). In contrast, interactions between CIAO3 and the CIA targeting complex were minimally affected by cellular iron levels (Fig. 2A). These results were validated by our targeted proteomics assay in which PRM was used to detect and quantify the levels of the CIA scaffold complex and the CIA targeting complex present in CIAO3 immunoprecipitates isolated from iron-replete and iron-depleted conditions. We confirmed that CIAO3 interacts with NUBP1 and NUBP2 in an iron-dependent manner and observed that both CIAO3–NUBP1 and CIAO3–NUBP2 interactions were reduced ∼4-fold in iron-deficient conditions indicating that CIAO3 dissociates from the intact CIA scaffold complex (Fig. 2B). Conversely, interactions between CIAO3 and the CIA targeting complex were only subtly influenced by iron levels and did not reach statistical significance (Fig. 2C). To validate that endogenous CIAO3 also interacts with the CIA scaffold complex in an iron-dependent manner, we treated cells expressing 3HA-3FLAG–tagged NUBP2 with FAC or deferoxamine mesylate, immunoprecipitated NUBP2 from whole cell lysate using anti-HA beads, and immunoblotted with CIAO3 antibodies. Our data show that the CIAO3–NUBP2 interaction is stabilized by the addition of iron and impaired when iron is depleted through chelation (Fig. 2D). We further characterized the association between CIAO3 and its interactors in response to alterations in intracellular iron levels after different time periods of treatment. We treated cells expressing 3HA-3FLAG-CIAO3 with FAC or DFO for either 3 or 8 h. We performed anti-HA immunoprecipitation followed by immunoblotting with indicated antibodies (Fig. 2E). Our data showed that the CIAO3–NUBP2 interaction increased after 3 h of FAC treatment but slightly declined by 8 h of treatment. Iron chelation caused a strong reduction in NUBP2 binding to CIAO3 as early as 3 h after treatment with DFO and extended up to at least 8 h after treatment. Ferritin levels, as expected, were gradually increasing over the same time period. These observations suggest that the response of the CIAO3–NUBP2 interaction to changes in iron levels is rapid. Given the observation that CIAO3's interactome was regulated by iron availability, we next examined whether these interactions were also influenced by other environmental stimuli. First, we treated cultured cells with ROS and examined the effects on the CIAO3 interactome. Tert-butyl hydroperoxide (tBHP) oxidizes glutathione and induces oxidative stress (25Trotta R.J. Sullivan S.G. Stern A. Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and ha.Biochem. J. 1983; 212: 759-772Crossref PubMed Scopus (139) Google Scholar). After cells were exposed to tBHP for 4 h, 3HA-3FLAG-tagged CIAO3 was immunoprecipitated from whole cell extracts and immunoblotted with antibodies against both the CIA scaffold complex and the CIA targeting complex. We observed a diminished CIAO3 interaction with the CIA scaffold complex under oxidative stress (Fig. 3A). A decreased interaction between CIAO3 and the CIA targeting complex was also observed but was comparatively modest under the same conditions. In addition to oxidative stress, we also manipulated oxygen tension and examined its effect on CIAO3 interactions. We immunoprecipitated 3HA-3FLAG-CIAO3 from extracts derived from cells cultured in either 21% O2 or 1% O2 and determined its interactions by immunoblotting with antibodies of NUBP2. HIF1α served as a positive control for hypoxia. We found that the CIAO3–NUBP2 interaction was stabilized in cells cultured in 1% O2 (Fig. 3B). Together these results suggest that CIAO3-containing complexes are sensitive to the redox status of the cell. The versatile nature of Fe-S clusters allows them to sense changes in both intracellular iron availability and the redox status of the cell (1Crack J.C. Green J. Thomson A.J. Le Brun N.E. Iron-sulfur cluster sensor-regulators.Curr. Opin. Chem. Biol. 2012; 16: 35-44Crossref PubMed Scopus (84) Google Scholar, 26Pellicer Martinez M.T. Crack J.C. Stewart M.Y. Bradley J.M. Svistunenko D.A. Johnston A.W. et al.Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.eLife. 2019; 8e47804Crossref PubMed Scopus (16) Google Scholar). As such, we hypothesized that the ability of these environmental changes to influence CIAO3 interactions might stem from effects on the Fe-S clusters bound to CIAO3 or other key components of this pathway. To test this possibility, we examined how disruption of Fe-S cluster biogenesis pathways affected the CIAO3 interactome. Previous studies have shown that cytosolic Fe-S cluster biogenesis mediated by the CIA pathway depends on mitochondrial Fe-S cluster biogenesis by the ISC pathway and that depleting the ISC scaffold protein, ISCU, leads to reduced iron incorporation and protein stability of both CIAO3 and CIA substrates (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar, 27Balk J. Pierik A.J. Netz D.J. Mühlenhoff U. Lill R. The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins.EMBO J. 2004; 23: 2105-2115Crossref PubMed Scopus (180) Google Scholar). We depleted ISCU1/2 from cells using RNAi and then induced expression of 3HA-3FLAG-CIAO3 which was stably expressed under the control of a doxycycline-inducible promoter. We observed a reduced amount of CIAO3 in the cells with silenced ISCU1/2, which is consistent with previous observations (Fig. 3C) (2Stehling O. Vashisht A.A. Mascarenhas J. Jonsson Z.O. Sharma T. Netz D.J. et al.MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity.Science. 2012; 337: 195-199Crossref PubMed Scopus (226) Google Scholar). Immunoblots of the affinity-purified CIAO3 complexes showed reduced co-precipitation for both the CIA scaffold complex and the CIA targeting complex. Densitometric evaluation of CIAO3 interactions as shown in Figure 3C revealed >60% and >90% reduction in the amount of NUBP1 and NUBP2 co-purifying with CIAO3 in response to the silencing of ISCU1/2 (Fig. 3D). In addition, we observed that the interactions between CIAO3 and components of the CIA targeting complex also drastically diminished upon knockdown of ISCU1/2 (Fig. 3E). Together, our observations demonstrate the assembly of CIAO3 into higher order complexes depends on the presence of a functional Fe-S cluster biogenesis pathway. CIAO3, which plays an essential role in bridging early and late CIA steps, has two Fe-S cluster binding sites: one at its N terminus and the other at its C terminus (28Maione V. Grifagni D. Torricella F. Cantini F. Banci L. CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron–sulfur cluster assembly machinery.J. Biol. Inorg. Chem. 2020; 25: 501-508Crossref PubMed Scopus (5) Google Scholar, 29Urzica E. Pierik A.J. Mühlenhoff U. Lill R. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.Biochemistry. 2009; 48: 4946-4958Crossref PubMed Scopus (42) Google Scholar). Given that Fe-S clusters are intrinsically sensitive to the cellular environment and regulate the stability and/or function of Fe-S proteins, we reasoned that CIAO3 interactions may be regulated by its cluster incorporation status. Previous studies have indicated that missense mutations of CIAO3 substituting cysteine with serine at position 71 in the N terminus or at both positions 190 and 395 in the C terminus render the protein defective in binding of Fe-S clusters (28Maione V. Grifagni D. Torricella F. Cantini F. Banci L. CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron–sulfur cluster assembly machinery.J. Biol. Inorg. Chem. 2020; 25: 501-508Crossref PubMed Scopus (5) Google Scholar, 29Urzica E. Pierik A.J. Mühlenhoff U. Lill R. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.Biochemistry. 2009; 48: 4946-4958Crossref PubMed Scopus (42) Google Scholar). Based on these studies, we generated CIAO3 mutants with impaired cluster incorporation (C71S, C190S/C395S, and C71S/C190S/C395S) to determine whether the Fe-S cluster requirement observed for CIAO3 interactions was dependent on cluster binding by CIAO3 itself (Fig. 4A). To compare and quantify the interactomes of wildtype and mutant versions of CIAO3 with known CIA components, we utilized our PRM-based targeted proteomics assay that monitors known CIA components and substrates as described earlier. We purified both wildtype and mutant 3HA-3FLAG CIAO3 complexes and quantified their interactions with the CIA scaffold complex, the CIA targeting complex, and CIA substrates after normalization to the amount of CIAO3 present in each purification. We observed that CIAO3-NUBP1/2 interactions were dramatically reduced for more than 32-fold, consistent with our earlier observation that Fe-S clusters are required for the interaction between CIAO3 and the CIA scaffold complex (Fig. 4B). The CIA targeting complex also showed modestly reduced association with CIAO3 displaying an approximately 5-fol
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