Affinage

ISCA1

Iron-sulfur cluster assembly 1 homolog, mitochondrial · UniProt Q9BUE6

Length
129 aa
Mass
14.2 kDa
Annotated
2026-04-28
34 papers in source corpus 17 papers cited in narrative 17 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ISCA1 is a mitochondrial iron-sulfur cluster assembly protein that functions as the central organizer of the late-acting [4Fe-4S] biogenesis machinery. It receives [2Fe-2S] clusters from GLRX5 and, together with ISCA2 and IBA57, catalyzes their FDX2-dependent reductive fusion into [4Fe-4S] clusters on the ISCA1–ISCA2 heterodimer; ISCA1 then bridges ISCA2 and NFU1 in a transient ternary complex to drive [4Fe-4S] cluster transfer to NFU1 and ultimately to target apoproteins including aconitase, respiratory complex I, and lipoic acid synthase (PMID:32817474, PMID:33711344, PMID:22323289). Three conserved cysteine residues coordinate its [2Fe-2S] cluster, whose integrity is essential for function, and pathogenic variants that destabilize this cluster or impair mitochondrial import cause multiple mitochondrial dysfunctions syndrome type 5 (MMDS5) (PMID:10805735, PMID:32092383, PMID:29767723). Complete loss of ISCA1 in rats is embryonically lethal, and neuron-specific deletion recapitulates MMDS5 with epilepsy, mitochondrial fragmentation, and respiratory chain failure (PMID:31016283, PMID:37140997).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2000 High

    Initial characterization established that the ISCA1 ortholog (yeast Isa1) localizes to the mitochondrial matrix, requires three conserved cysteines for function, and is needed for activity of [4Fe-4S]-containing enzymes aconitase and succinate dehydrogenase — linking the protein to mitochondrial iron-sulfur metabolism.

    Evidence Genetic deletion, site-directed mutagenesis, subcellular fractionation, and enzyme activity assays in S. cerevisiae

    PMID:10805735

    Open questions at the time
    • Iron-sulfur cluster type bound by Isa1 not yet determined
    • Relationship to other known Fe/S assembly factors unclear
    • Mammalian function not tested
  2. 2002 High

    Spectroscopic reconstitution showed that Isa1 itself carries a [2Fe-2S] cluster and physically associates with ferredoxin, providing the first direct evidence that the protein is an Fe/S-binding factor rather than simply an iron donor.

    Evidence Mössbauer, UV-visible, and EPR spectroscopy with chemical crosslinking and mutagenesis on fission yeast Isa1

    PMID:11941510

    Open questions at the time
    • Role of the ferredoxin interaction in cluster chemistry unknown
    • Whether the protein can also bind mononuclear iron unclear
  3. 2004 Medium

    Human ISCA1 was shown to localize to mitochondria and functionally replace yeast Isa1, establishing evolutionary conservation and validating the yeast model for human studies.

    Evidence Yeast complementation and subcellular localization of human ISCA1

    PMID:15262227

    Open questions at the time
    • Human-specific interaction partners not identified
    • No human loss-of-function data
  4. 2007 High

    Genetic epistasis revealed that Isa1/Isa2 are specifically required for functional maturation of mitochondrial [4Fe-4S] proteins but not for de novo Fe/S cluster synthesis on the scaffold protein Isu1/Isu2, placing the ISA complex downstream in a dedicated [4Fe-4S] branch.

    Evidence Genetic epistasis with Isu1/Isu2 and Isa1/Isa2 depletions, biotin synthase activity as [4Fe-4S] reporter in yeast

    PMID:17259550

    Open questions at the time
    • Biochemical mechanism of [4Fe-4S] cluster formation not reconstituted
    • Upstream [2Fe-2S] donor to Isa not identified
  5. 2010 High

    The upstream [2Fe-2S] donor GLRX5 was linked to ISCA1/ISCA2, and ISCA1 was shown to bind mononuclear iron with very high affinity and donate it for Fe/S assembly, raising the question of whether its primary role is as an iron chaperone or cluster relay.

    Evidence BiFC interaction assay and genetic suppression in fission yeast (GLRX5); iron-binding and iron-transfer assays with purified human ISCA1 in vitro

    PMID:20085751 PMID:20302570

    Open questions at the time
    • Whether iron or [2Fe-2S] cluster donation is the physiological function debated
    • Full reconstitution of [4Fe-4S] formation from physiological components not achieved
  6. 2011 High

    Comprehensive in vivo analysis confirmed that the Isa1–Isa2 complex is selectively required for mitochondrial [4Fe-4S] but not [2Fe-2S] proteins, and that the partner IBA57 is needed for the same step, defining the three-component late-acting [4Fe-4S] assembly system.

    Evidence In vivo reporters for [2Fe-2S] and [4Fe-4S] protein function, iron-binding assays, and IBA57 depletion in yeast

    PMID:21987576

    Open questions at the time
    • Mechanism of IBA57 contribution unknown
    • How [2Fe-2S] clusters convert to [4Fe-4S] not resolved
  7. 2012 High

    Studies in human cells confirmed that ISCA1, ISCA2, and IBA57 are specifically required for mitochondrial [4Fe-4S] proteins including respiratory complex I and lipoic acid synthase, refuting an earlier claim of cytosolic function and showing that their loss causes severe mitochondrial structural damage.

    Evidence RNAi knockdown in HeLa cells with enzyme assays, electron microscopy, and subcellular fractionation

    PMID:22323289

    Open questions at the time
    • Direct protein–protein interactions among the three factors not mapped
    • Downstream transfer route to target apoproteins unknown
  8. 2018 High

    A pathogenic ISCA1 missense mutation (p.V10G) was shown to impair mitochondrial import and protein stability, directly linking ISCA1 loss of function to human disease (MMDS5) with defective [4Fe-4S] protein maturation.

    Evidence Patient fibroblast biochemistry, mitochondrial import assay, RNAi complementation with wild-type vs. mutant ISCA1

    PMID:29767723

    Open questions at the time
    • Structural basis for import defect not determined
    • Genotype-phenotype correlations across ISCA1 variants not established
  9. 2019 Medium

    Complete and neuron-specific knockout of ISCA1 in rats established that the gene is essential for embryonic viability and neuronal mitochondrial integrity, with neuron-specific loss recapitulating MMDS5 features including epilepsy and respiratory chain failure.

    Evidence CRISPR-Cas9 whole-body and conditional (NeuN-Cre) knockout rats with phenotypic, ultrastructural, and biochemical analysis

    PMID:31016283 PMID:37140997

    Open questions at the time
    • Cell-type-specific vulnerability beyond neurons not tested
    • Whether compensatory pathways exist in other tissues unknown
  10. 2020 High

    Full in vitro reconstitution resolved the complete [4Fe-4S] assembly mechanism: GLRX5 donates [2Fe-2S] clusters to ISCA1–ISCA2, FDX2 (not FDX1) provides the electrons for reductive [2Fe-2S]-to-[4Fe-4S] fusion, and ISCA1 then transfers the [4Fe-4S] cluster to NFU1 via a direct interaction mapped to NFU1's C-terminal hydrophobic patch.

    Evidence Reconstitution of [4Fe-4S] aconitase maturation with purified human proteins, spectroscopy, Co-IP, and NFU1 mutagenesis

    PMID:32776106 PMID:32817474

    Open questions at the time
    • Structural model of the ISCA1–ISCA2 heterodimer with bound [4Fe-4S] cluster lacking
    • How IBA57 participates mechanistically in the fusion reaction unresolved
  11. 2021 High

    NMR-based interaction mapping established ISCA1 as the central organizer: it bridges ISCA2 and NFU1 (which do not interact directly) in a transient ternary complex, driving directional [4Fe-4S] cluster transfer from the ISCA1–ISCA2 assembly site to an ISCA1–NFU1 delivery site.

    Evidence NMR spectroscopy, protein interaction mapping, ternary complex characterization, cluster transfer monitoring with purified human proteins

    PMID:33711344

    Open questions at the time
    • Atomic-resolution structure of the ternary complex not available
    • Kinetics and regulation of cluster transfer in vivo unknown
    • How target apoprotein specificity is achieved downstream of NFU1 not determined
  12. 2023 Medium

    ISCA1 was found to bind copper with high affinity, and copper excess inhibits Fe/S cluster assembly by competing at ISCA1/ISCA2/ISCU, providing a molecular basis for copper cytotoxicity in Wilson's disease.

    Evidence Copper-binding assays with purified proteins, Fe/S enzyme activity in ATP7B-knockdown cells and ATP7A-deficient lymphocytes, Wilson's disease mouse model

    PMID:37225108

    Open questions at the time
    • Structural basis of copper vs. iron competition not determined
    • In vivo contribution of ISCA1 copper binding relative to other targets unclear

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the atomic structure of the ISCA1–ISCA2 heterodimer with bound [4Fe-4S] cluster, the precise catalytic role of IBA57 in the reductive fusion reaction, how the assembled [4Fe-4S] clusters are directed from NFU1 to specific target apoproteins, and the regulatory mechanisms controlling ISCA1 activity in different tissues.
  • No high-resolution structure of the ISCA1–ISCA2–[4Fe-4S] complex
  • IBA57 mechanism of action unresolved
  • Target apoprotein selectivity mechanism unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140104 molecular carrier activity 4 GO:0016740 transferase activity 3
Localization
GO:0005739 mitochondrion 4
Pathway
R-HSA-1430728 Metabolism 5 R-HSA-1643685 Disease 2
Partners
FDX2GLRX5IBA57ISCA2ISCU2NFU1
Complex memberships
ISCA1-ISCA2 heterodimerISCA1-ISCA2-IBA57 complexISCA1-ISCA2-NFU1 ternary complex

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2000 Yeast Isa1p (ISCA1 ortholog) is targeted to the mitochondrial matrix, and its three invariant cysteine residues are essential for function, likely involved in iron binding; loss of Isa1p causes reduced aconitase and succinate dehydrogenase activity and elevated mitochondrial iron accumulation. Genetic deletion, subcellular fractionation, site-directed mutagenesis of cysteine residues, enzyme activity assays Molecular and cellular biology High 10805735
2002 Fission yeast Isa1 (ISCA1 ortholog) is a multimeric protein carrying [2Fe-2S]2+ clusters; it forms a complex with a redox-active ferredoxin (identified by crosslinking), and cysteine ligands are required for cluster coordination as shown by site-directed mutagenesis. Mössbauer spectroscopy, UV-visible and EPR spectroscopy, chemical crosslinking, site-directed mutagenesis Journal of biological inorganic chemistry High 11941510
2004 Human ISCA1 (hIscA) is localized to mitochondria and functionally complements yeast isa1Δ mutants, establishing functional conservation; the protein contains the HESB structural domain. Yeast complementation assay, subcellular localization (mitochondrial targeting), immunological detection Biochimica et biophysica acta Medium 15262227
2007 Yeast Isa1 and Isa2 are required for the in vivo catalytic function of biotin synthase (Bio2), a [4Fe-4S] enzyme, but not for the de novo synthesis of its Fe/S clusters (which depends on Isu1/Isu2); Isa proteins are specifically needed for functional activity of [4Fe-4S] apoproteins. Genetic epistasis, enzyme activity assays, protein depletion, overexpression rescue Eukaryotic cell High 17259550
2009 Human ISCA1 is present in both cytosolic and mitochondrial fractions, and interacts with IOP1/NARFL (a cytosolic CIA pathway factor); siRNA knockdown of ISCA1 in HeLa cells reduces activity of both mitochondrial (aconitase, succinate dehydrogenase) and cytosolic (cytosolic aconitase) Fe/S enzymes. Subcellular fractionation, Co-immunoprecipitation, siRNA knockdown, enzyme activity assays The Journal of biological chemistry Medium 19864422
2010 Human ISCA1 (hIscA1) binds mononuclear iron with an association constant of ~2×10^19 M^-1, and iron-bound hIscA1 can donate iron for Fe/S cluster assembly on the E. coli scaffold protein IscU in vitro, supporting an iron chaperone role. UV-visible absorption, EPR spectroscopy, in vitro iron transfer assay to IscU, iron content modulation by growth medium The Biochemical journal High 20302570
2010 In fission yeast, Grx5 physically interacts in vivo with Isa1 and Isa2 in mitochondria (bimolecular fluorescence complementation); overexpression of isa1+ or isa2+ suppresses growth defects of Δgrx5 mutant, placing Grx5 upstream or in the same pathway as Isa1/Isa2 in Fe/S assembly. Bimolecular fluorescence complementation (BiFC), genetic suppressor screen, multi-copy suppression, Fe/S enzyme activity assays Biochemical and biophysical research communications Medium 20085751
2011 Yeast Isa1 and Isa2 form a complex specifically required for maturation of mitochondrial [4Fe-4S] proteins (aconitase, homoaconitase) but dispensable for [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins; Isa1/Isa2 bind iron in vivo, and this iron is proposed as the direct donor for de novo [4Fe-4S] cluster synthesis in a reaction assisted by Iba57. In vivo genetic analysis, iron-binding assay, targeted mitochondrial [2Fe-2S]/[4Fe-4S] ferredoxin reporters, Iba57 depletion, enzyme activity assays The Journal of biological chemistry High 21987576
2012 Human ISCA1, ISCA2, and IBA57 are specifically required for maturation of mitochondrial [4Fe-4S] proteins (aconitase, respiratory complex I, lipoic acid synthase) but not [2Fe-2S] proteins (ferrochelatase); ISCA1/ISCA2 do not function in the cytosol for Fe/S protein maturation; their depletion causes massive mitochondrial swelling and loss of cristae. RNAi knockdown in HeLa cells, enzyme activity assays, heme quantification, mitochondrial morphology by electron microscopy, subcellular fractionation Molecular biology of the cell High 22323289
2018 A homozygous missense mutation p.(V10G) in the ISCA1 presequence severely impairs mitochondrial import and stability of ISCA1 protein, leading to defective [4Fe-4S] protein maturation (respiratory complexes, lipoic acid synthesis); RNAi knockdown rescued by wild-type but not mutant ISCA1. Patient fibroblast biochemical analysis, mitochondrial import assay, RNAi complementation, targeted MitoExome sequencing Human molecular genetics High 29767723
2020 Mitochondrial [4Fe-4S] cluster assembly on ISCA1-ISCA2 proceeds by reductive fusion of two [2Fe-2S] clusters donated from GLRX5; this reaction requires FDX2 (ferredoxin 2) as the specific electron donor and its reductase FDXR, as well as IBA57, reconstituted in vitro without artificial reductants. FDX1 and other reducing systems cannot substitute for FDX2. In vitro reconstitution of [4Fe-4S] aconitase maturation with purified components, spectroscopy, functional specificity tests with FDX1 vs FDX2 Proceedings of the National Academy of Sciences of the United States of America High 32817474
2020 ISCA1 directly interacts with both ISCU2 and NFU1, donating [2Fe-2S] clusters to NFU1 (along with ISCU2) for reductive formation of a bridging [4Fe-4S] cluster; the interaction site maps to a conserved hydrophobic patch on the C-terminal alpha-helix of NFU1, and mutagenesis of this site abolishes cluster acquisition. Co-immunoprecipitation, biochemical reconstitution, site-directed mutagenesis of NFU1 interaction surface, functional assays of downstream targets (lipoic acid synthase, pyruvate dehydrogenase lipoylation) Human molecular genetics High 32776106
2020 A novel pathogenic ISCA1 variant p.(Tyr101Cys) decreases stability of the [2Fe-2S] cluster bound to the protein, establishing that ISCA1 itself carries a [2Fe-2S] cluster whose integrity is essential for its function in [4Fe-4S] protein maturation. Expression and purification of recombinant mutant ISCA1, spectroscopic characterization of [2Fe-2S] cluster stability, patient fibroblast biochemistry Mitochondrion Medium 32092383
2021 ISCA1 acts as the central organizer of the late-acting [4Fe-4S] assembly complex: it interacts with both ISCA2 and NFU1 individually but ISCA2 and NFU1 do not interact with each other; ISCA1 promotes formation of a transient ISCA1-ISCA2-NFU1 ternary complex and drives [4Fe-4S] cluster transfer from the ISCA1-ISCA2 assembly site to a cluster-binding site formed by ISCA1 and NFU1. NMR spectroscopy, protein interaction mapping, ternary complex characterization, cluster transfer monitoring Journal of molecular biology High 33711344
2019 Knockout of ISCA1 in rats causes early embryonic lethality (death by embryonic day 8.5), with decreased NDUFA9 (complex I subunit) protein levels and increased aconitase 2 protein in embryos, demonstrating an essential in vivo role in mitochondrial respiratory chain assembly. CRISPR-Cas9 knockout rats, Western blot of mitochondrial proteins, embryonic phenotype analysis Animal models and experimental medicine Medium 31016283
2023 Human ISCA1, ISCA2, and ISCU proteins have strong copper-binding activity, and excess copper inhibits Fe/S cluster assembly by competing at these proteins, providing a mechanism for copper cytotoxicity in Wilson's disease. Copper-binding assay with purified proteins, Fe/S enzyme activity assays in cells and mouse model of Wilson's disease (ATP7B knockdown, ATP7A-/- lymphocytes), in vivo and in vitro pharmacological analysis Free radical biology & medicine Medium 37225108
2023 Neuron-specific Isca1 knockout rats develop MMDS5-like disease with epilepsy, memory impairment, neuronal death, mitochondrial fragmentation and cristae rupture, reduced respiratory chain complex proteins, and decreased ATP production, demonstrating that ISCA1 is required for neuronal mitochondrial integrity. Conditional CRISPR-Cas9 knockout (Isca1flox/flox-NeuN-Cre), MRI, behavioral testing, TEM, Western blot, ATP assay, immunofluorescence Animal models and experimental medicine Medium 37140997

Source papers

Stage 0 corpus · 34 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2012 The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation. Molecular biology of the cell 171 22323289
2000 Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis. Molecular and cellular biology 148 10805735
2011 Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins. The Journal of biological chemistry 135 21987576
2011 Functional diversity of isoamylase oligomers: the ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm. Plant physiology 73 21436381
2020 Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2. Proceedings of the National Academy of Sciences of the United States of America 69 32817474
2002 Iron-sulfur cluster biosynthesis: characterization of Schizosaccharomyces pombe Isa1. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 68 11941510
2018 Endosperm sugar accumulation caused by mutation of PHS8/ISA1 leads to pre-harvest sprouting in rice. The Plant journal : for cell and molecular biology 62 29775500
2010 Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast. Biochemical and biophysical research communications 53 20085751
2017 Homozygous p.(Glu87Lys) variant in ISCA1 is associated with a multiple mitochondrial dysfunctions syndrome. Journal of human genetics 52 28356563
2007 The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae. Eukaryotic cell 47 17259550
2014 Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly. The Journal of biological chemistry 46 24993830
2009 Human ISCA1 interacts with IOP1/NARFL and functions in both cytosolic and mitochondrial iron-sulfur protein biogenesis. The Journal of biological chemistry 41 19864422
2011 Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues. Molecular microbiology 33 21790804
2010 Iron-binding activity of human iron-sulfur cluster assembly protein hIscA1. The Biochemical journal 32 20302570
2023 Copper exerts cytotoxicity through inhibition of iron-sulfur cluster biogenesis on ISCA1/ISCA2/ISCU assembly proteins. Free radical biology & medicine 31 37225108
2004 hIscA: a protein implicated in the biogenesis of iron-sulfur clusters. Biochimica et biophysica acta 29 15262227
2018 ISCA1 mutation in a patient with infantile-onset leukodystrophy causes defects in mitochondrial [4Fe-4S] proteins. Human molecular genetics 25 29767723
2014 Replacement of the endogenous starch debranching enzymes ISA1 and ISA2 of Arabidopsis with the rice orthologs reveals a degree of functional conservation during starch synthesis. PloS one 21 24642810
2020 Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1. Human molecular genetics 20 32776106
2013 Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf. The New phytologist 20 23952574
2017 Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype. PloS one 19 28708852
2019 Knockout of ISCA1 causes early embryonic death in rats. Animal models and experimental medicine 13 31016283
2023 Intermittent Theta Burst Stimulation Attenuates Cognitive Deficits and Alzheimer's Disease-Type Pathologies via ISCA1-Mediated Mitochondrial Modulation in APP/PS1 Mice. Neuroscience bulletin 11 37578635
2021 ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins. Journal of molecular biology 11 33711344
2022 Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice. Plant molecular biology 10 35083581
2018 Report of the Third Family with Multiple Mitochondrial Dysfunctions Syndrome 5 Caused by the Founder Variant p.(Glu87Lys) in ISCA1. Journal of pediatric genetics 8 30105122
2017 Molecular characterization, spatial-temporal expression and magnetic response patterns of iron-sulfur cluster assembly1 (IscA1) in the rice planthopper, Nilaparvata lugens. Insect science 8 29063672
2023 A neuron-specific Isca1 knockout rat developments multiple mitochondrial dysfunction syndromes. Animal models and experimental medicine 6 37140997
2021 Transcriptomic analysis reveals the inhibition of reproduction in rice brown planthopper, Nilaparvata lugens, after silencing the gene of MagR (IscA1). Insect molecular biology 6 33410574
2020 Expanding the phenotype of mitochondrial disease: Novel pathogenic variant in ISCA1 leading to instability of the iron-sulfur cluster in the protein. Mitochondrion 6 32092383
2025 Amylopectin branch trimming and biosynthesis elucidated by the rice isoamylase ISA1-ISA2 heterocomplex. Nature communications 4 40595605
2022 Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype. International journal of molecular sciences 4 36613634
2025 Long Noncoding RNA ISA1 Protects Against Ischemic Brain Damage by Promoting the Transformation of Microglia Toward Anti-inflammatory Phenotype via the SOCS3/JAK2/STAT3 Pathway. Neurochemical research 1 39891829
2024 Comprehensive Analysis Reveals That ISCA1 Is Correlated with Ferroptosis-Related Genes Across Cancers and Is a Biomarker in Thyroid Carcinoma. Genes 0 39766805