COX4I1

---
id: P13073
gene_symbol: COX4I1
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  Cytochrome c oxidase subunit 4 isoform 1 (COX4-1), the ubiquitously expressed
  nuclear-encoded regulatory subunit of mitochondrial Complex IV (cytochrome c
  oxidase). COX4-1 is a single-pass transmembrane protein of the inner
  mitochondrial membrane with a large matrix-facing regulatory domain and a
  C-terminal intermembrane-space segment that contacts COX2 to help shape the
  cytochrome c docking site. Although noncatalytic, COX4-1 is the primary
  regulatory subunit of Complex IV, carrying an allosteric ATP/ADP-binding site
  in its matrix domain that mediates feedback inhibition of CIV activity at high
  ATP/ADP ratios. Phosphorylation of Ser58 by intramitochondrial PKA (via the
  sAC-cAMP-PKA axis) prevents ATP binding and relieves this inhibition, acting
  as a rapid bioenergetic switch. COX4-1 participates in early assembly modules
  (COX4-COX5A) and contributes to Complex IV stability and respirasome
  (CI-CIII2-CIV) formation. A tissue-specific isoform, COX4I2, is expressed
  under hypoxia and in lung/brain, and isoform switching between COX4-1 and
  COX4-2 modulates oxygen affinity and redox signaling. Pathogenic COX4I1
  variants (e.g., K101N, P152T) cause autosomal recessive Complex IV deficiency
  (MC4DN16) with growth failure, developmental regression, and chromosomal
  instability.
existing_annotations:
# ============================================================
# CC: Cellular Component annotations
# ============================================================
  - term:
      id: GO:0045277
      label: respiratory chain complex IV
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        COX4I1 is a bona fide subunit of Complex IV (cytochrome c oxidase).
        Structural studies confirm it is one of 14 subunits of the intact
        monomeric CIV (PMID:30030519). This IBA annotation is well supported
        by phylogenetic inference across eukaryotes and by direct structural
        and biochemical evidence.
      action: ACCEPT
      reason: >-
        Core component annotation. COX4I1 is one of the 14 subunits of Complex IV,
        confirmed by cryo-EM structure of the intact human CIV (PMID:30030519) and
        UniProt which lists it as "Component of the cytochrome c oxidase (complex
        IV, CIV), a multisubunit enzyme composed of 14 subunits." Phylogenetic
        inference (IBA) is entirely consistent.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: >-
            we obtained the entire CIV structure containing 14 subunits, which
            includes the extra subunit NDUFA4
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            Component of the cytochrome c oxidase (complex IV, CIV), a multisubunit
            enzyme composed of 14 subunits. The complex is composed of a catalytic
            core of 3 subunits MT-CO1, MT-CO2 and MT-CO3, encoded in the
            mitochondrial DNA, and 11 supernumerary subunits COX4I1 (or COX4I2),
            COX5A, COX5B, COX6A1 (or COX6A2), COX6B1 (or COX6B2), COX6C, COX7A2
            (or COX7A1), COX7B, COX7C, COX8A and COXFA4
  - term:
      id: GO:0006123
      label: mitochondrial electron transport, cytochrome c to oxygen
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        COX4I1, as a subunit of Complex IV, is involved in the process of
        electron transport from cytochrome c to oxygen. While COX4I1 does not
        directly participate in electron transfer (catalyzed by the mtDNA-encoded
        core subunits COX1/2/3), it is required for complex assembly, stability,
        and allosteric regulation of this process (PMID:30030519). IBA inference
        is well supported.
      action: ACCEPT
      reason: >-
        Core biological process annotation. COX4I1 is essential for Complex IV
        function. Pathogenic variants in COX4I1 cause Complex IV deficiency
        (PMID:28766551), demonstrating it is required for electron transport from
        cytochrome c to oxygen. The IBA annotation is phylogenetically sound.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: >-
            CIV is the terminal oxidase of the electron transport chain in
            mitochondria...It accepts electrons from cytochrome c to reduce the
            oxygen to water and meanwhile pumps two protons from the matrix side
            to the intermembrane space (IMS)
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            Cytochrome c oxidase is the component of the respiratory chain that
            catalyzes the reduction of oxygen to water. Electrons originating from
            reduced cytochrome c in the intermembrane space (IMS) are transferred
            via the dinuclear copper A center (CU(A)) of subunit 2 and heme A of
            subunit 1 to the active site in subunit 1
  - term:
      id: GO:1902600
      label: proton transmembrane transport
    evidence_type: IEA
    original_reference_id: GO_REF:0000108
    review:
      summary: >-
        Complex IV couples electron transfer to proton pumping across the inner
        mitochondrial membrane. As a subunit of CIV, COX4I1 contributes to this
        process. The IEA annotation is derived from the logical inference that
        cytochrome-c oxidase activity (GO:0004129) implies proton transmembrane
        transport.
      action: ACCEPT
      reason: >-
        Acceptable IEA inference. Complex IV pumps protons during electron
        transport, and COX4I1 as a structural/regulatory subunit is involved
        in this process. While COX4I1 does not directly form the proton channel,
        the annotation captures the broader process the complex participates in.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: >-
            It accepts electrons from cytochrome c to reduce the oxygen to water
            and meanwhile pumps two protons from the matrix side to the
            intermembrane space (IMS)
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        COX4I1 is a single-pass transmembrane protein of the mitochondrial inner
        membrane. This is confirmed by cryo-EM structural data (PMID:30030519)
        and UniProt annotation.
      action: ACCEPT
      reason: >-
        Core localization confirmed by structural studies and multiple lines of
        evidence. COX4I1 has a transmembrane helix (residues 99-124) spanning
        the inner mitochondrial membrane, with a matrix-facing regulatory domain
        and an IMS-facing C-terminal segment.
      supported_by:
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            SUBCELLULAR LOCATION: Mitochondrion inner membrane...Single-pass
            membrane protein
        - reference_id: PMID:30030519
          supporting_text: >-
            we obtained the entire CIV structure containing 14 subunits
  - term:
      id: GO:0005758
      label: mitochondrial intermembrane space
    evidence_type: IEA
    original_reference_id: GO_REF:0000117
    review:
      summary: >-
        COX4I1 has a C-terminal segment (residues 125-169) that faces the
        intermembrane space, contacting COX2 to shape the cytochrome c docking
        site. However, COX4I1 is primarily an inner membrane protein with matrix
        and IMS domains, not a soluble IMS protein. This annotation is somewhat
        misleading as a localization statement.
      action: ACCEPT
      reason: >-
        While COX4I1 is not a soluble IMS protein, it does have a topology domain
        in the intermembrane space (residues 125-169), making this annotation
        technically correct. UniProt topology annotation confirms this. The IEA
        prediction is consistent with the known topology.
      supported_by:
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            TOPO_DOM 125..169 /note="Mitochondrial intermembrane"
  - term:
      id: GO:0006123
      label: mitochondrial electron transport, cytochrome c to oxygen
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: >-
        Duplicate of the IBA annotation for the same GO term. This IEA annotation
        is based on InterPro domain mapping (IPR004203, IPR036639) to the COX
        subunit IV family. Consistent with the IBA annotation above.
      action: ACCEPT
      reason: >-
        Redundant with IBA annotation but correctly inferred from domain
        composition. The COX4 domain family (Pfam PF02936) is specific to
        cytochrome c oxidase subunit IV, making this IEA inference sound.
  - term:
      id: GO:0045277
      label: respiratory chain complex IV
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        Duplicate of the IBA annotation for the same GO term. This IEA annotation
        is from combined automated methods. Consistent with structural and
        phylogenetic evidence.
      action: ACCEPT
      reason: >-
        Redundant with the IBA annotation but correctly inferred. COX4I1 is a
        well-established subunit of Complex IV.

# ============================================================
# MF: Molecular Function annotations - protein binding
# ============================================================
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:23260140
    review:
      summary: >-
        PMID:23260140 (Mick et al. 2012, Cell) describes MITRAC complexes - early
        cytochrome c oxidase assembly intermediates. COX4I1 was found as a
        component of MITRAC complexes, interacting with MT-CO1 (UniProtKB:P00395).
        The WITH/FROM column shows the interactor is MT-CO1. This interaction
        represents a CIV assembly intermediate interaction between a nuclear-
        encoded subunit and the catalytic core subunit.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        The interaction between COX4I1 and MT-CO1 is a subunit-subunit interaction
        within Complex IV and during CIV assembly. Annotating this as generic
        "protein binding" is uninformative. The interaction is better captured by
        the part_of GO:0045277 (respiratory chain complex IV) annotation and could
        be more specifically annotated as part of Complex IV assembly.
      supported_by:
        - reference_id: PMID:23260140
          supporting_text: >-
            we report a comprehensive dissection of early cytochrome c oxidase
            assembly intermediates containing proteins required for normal
            mitochondrial translation and reveal assembly factors promoting
            biogenesis of human respiratory-chain complexes
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:25416956
    review:
      summary: >-
        PMID:25416956 (Rolland et al. 2014, Cell) is a large-scale proteome-wide
        Y2H interactome study. The WITH/FROM shows the interactor is SDCBP
        (O00560, syntenin-1). This is a high-throughput interaction without
        targeted validation for COX4I1 specifically.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Generic "protein binding" from a large-scale Y2H screen. SDCBP (syntenin-1)
        is a PDZ domain-containing scaffolding protein primarily associated with
        cell surface signaling and exosomes. An interaction between the
        mitochondrial inner membrane protein COX4I1 and the cytoplasmic/membrane
        protein SDCBP is of uncertain biological significance and likely reflects
        the high false-positive rate of Y2H screens for proteins in different
        compartments.
      supported_by:
        - reference_id: PMID:25416956
          supporting_text: >-
            Here, we describe a systematic map of ?14,000 high-quality human
            binary protein-protein interactions
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:28514442
    review:
      summary: >-
        PMID:28514442 (Huttlin et al. 2017, Nature) is the BioPlex 2.0
        large-scale AP-MS interactome study. The WITH/FROM shows the interactor
        is DBT (P11182, dihydrolipoamide branched chain transacylase). Both
        proteins are mitochondrial matrix-associated, so the interaction is at
        least compartment-consistent.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Generic "protein binding" from a large-scale AP-MS screen. While both
        COX4I1 and DBT are mitochondrial proteins, DBT is a component of the
        branched-chain alpha-keto acid dehydrogenase complex, not Complex IV.
        The interaction may reflect co-purification from mitochondrial extracts
        rather than a specific functional interaction. "Protein binding" is
        uninformative regardless.
      supported_by:
        - reference_id: PMID:28514442
          supporting_text: >-
            Here we present BioPlex 2.0 (Biophysical Interactions of
            ORFeome-derived complexes), which uses robust affinity
            purification-mass spectrometry methodology to elucidate protein
            interaction networks and co-complexes nucleated by more than 25% of
            protein-coding genes from the human genome
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:31515488
    review:
      summary: >-
        PMID:31515488 (Fragoza et al. 2019, Nat Commun) is a large-scale study
        of how genetic variants disrupt protein-protein interactions. The
        WITH/FROM shows interaction with SDCBP (O00560). Same interactor as the
        PMID:25416956 annotation above. This study primarily characterizes how
        SNVs affect known interactions from Y2H.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Same reasoning as for PMID:25416956 - this is the same COX4I1-SDCBP
        interaction from Y2H screening. Generic "protein binding" is
        uninformative, and the biological relevance of a mitochondrial inner
        membrane protein interacting with the cytoplasmic scaffolding protein
        SDCBP is questionable.
      supported_by:
        - reference_id: PMID:31515488
          supporting_text: Extensive disruption of protein interactions by genetic
            variants across the allele frequency spectrum in human populations.
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:32814053
    review:
      summary: >-
        PMID:32814053 (Haenig et al. 2020, Cell Rep) is a neurodegenerative
        disease interactome mapping study. The WITH/FROM shows the interactor is
        KLF11 (O14901, Krueppel-like factor 11), a nuclear transcription factor.
        An interaction between a mitochondrial inner membrane protein and a
        nuclear transcription factor is biologically implausible.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Generic "protein binding" from a disease-focused interactome screen.
        KLF11 is a nuclear Krueppel-like transcription factor involved in TGF-beta
        signaling. A direct physical interaction between COX4I1 (mitochondrial
        inner membrane) and KLF11 (nucleus) is not biologically plausible and
        likely represents a false positive from the high-throughput screen.
      supported_by:
        - reference_id: PMID:32814053
          supporting_text: Interactome Mapping Provides a Network of Neurodegenerative
            Disease Proteins and Uncovers Widespread Protein Aggregation in Affected
            Brains.
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:33961781
    review:
      summary: >-
        PMID:33961781 (Huttlin et al. 2021, Cell) is the BioPlex 3.0 dual
        proteome-scale AP-MS interactome study. The GOA file shows two entries
        for this PMID - one with MT-CO1 (P00395) and one with DBT (P11182).
        The MT-CO1 interaction represents a genuine intra-complex interaction,
        while the DBT interaction is likely co-purification.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Generic "protein binding" is uninformative even when the interaction
        (with MT-CO1) is biologically meaningful. The COX4I1-MT-CO1 interaction
        is a direct subunit-subunit contact within Complex IV, already captured
        by the part_of GO:0045277 annotation. The DBT interaction is likely
        co-purification from mitochondrial extracts.
      supported_by:
        - reference_id: PMID:33961781
          supporting_text: >-
            Thousands of interactions assemble proteins into modules that impart
            spatial and functional organization to the cellular proteome
# Note: there are actually 2 lines in the GOA for PMID:33961781, one with MT-CO1 and one with DBT.
# The second one is captured here:
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:33961781
    review:
      summary: >-
        Second entry from PMID:33961781 BioPlex 3.0 study, with interactor
        DBT (P11182, dihydrolipoamide branched chain transacylase). See
        previous entry for same PMID for general assessment.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Same reasoning as above. Generic "protein binding" with DBT from
        large-scale AP-MS. DBT is a mitochondrial matrix enzyme of the BCKD
        complex, not a known functional partner of Complex IV. Likely
        co-purification artifact.
# ============================================================
# CC: Additional cellular component annotations
# ============================================================
      supported_by:
        - reference_id: PMID:33961781
          supporting_text: Dual proteome-scale networks reveal cell-specific remodeling
            of the human interactome.
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IEA
    original_reference_id: GO_REF:0000107
    review:
      summary: >-
        COX4I1 is imported to the mitochondrion where it is a subunit of Complex
        IV in the inner membrane. This broad localization is well supported by
        all evidence.
      action: ACCEPT
      reason: >-
        Correct IEA annotation. COX4I1 is a mitochondrial protein by all lines
        of evidence - structural, biochemical, and computational. While more
        specific terms (inner membrane, Complex IV) exist and are also annotated,
        the broader mitochondrion annotation is acceptable.
  - term:
      id: GO:0005829
      label: cytosol
    evidence_type: IEA
    original_reference_id: GO_REF:0000107
    review:
      summary: >-
        COX4I1 is synthesized in the cytosol as a precursor with a mitochondrial
        transit peptide (residues 1-22), which is cleaved upon import. The mature
        protein resides in the mitochondrial inner membrane. Cytosolic localization
        is only transient during biosynthesis.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        While COX4I1 precursor is transiently in the cytosol during translation
        and before mitochondrial import, the functional protein resides in the
        mitochondrial inner membrane. This IEA annotation likely comes from
        Ensembl Compara transfer from mouse data where cytosolic detection may
        reflect the precursor form. The cytosol is not the functional location
        of COX4I1.
  - term:
      id: GO:0007584
      label: response to nutrient
    evidence_type: IEA
    original_reference_id: GO_REF:0000107
    review:
      summary: >-
        This annotation is transferred from rat COX4I1 (P10888) via Ensembl
        Compara. COX4I1 does have an allosteric ATP/ADP regulatory site that
        senses energy status, but "response to nutrient" is too vague and
        indirect to describe this regulatory function.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        While COX4I1 activity is modulated by cellular energy status (ATP/ADP
        ratio) and could indirectly respond to nutrient availability, this term
        is too broad and non-specific. The primary regulatory function of COX4I1
        is allosteric inhibition by ATP, not a direct response to nutrients. This
        IEA transfer is over-annotation.
  - term:
      id: GO:0031966
      label: mitochondrial membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000107
    review:
      summary: >-
        COX4I1 is a mitochondrial inner membrane protein. The term "mitochondrial
        membrane" is a parent of "mitochondrial inner membrane" and is less
        specific but not wrong.
      action: ACCEPT
      reason: >-
        Correct but less specific than the available inner membrane annotations.
        This IEA is acceptable as a broader localization term that is consistent
        with the more specific inner membrane annotations.
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IDA
    original_reference_id: GO_REF:0000052
    review:
      summary: >-
        IDA annotation based on curation of immunofluorescence data (HPA).
        COX4I1 is detected in mitochondria by immunofluorescence in the Human
        Protein Atlas.
      action: ACCEPT
      reason: >-
        Direct experimental evidence (immunofluorescence) confirming
        mitochondrial localization. Consistent with all other localization
        evidence.
  - term:
      id: GO:0006123
      label: mitochondrial electron transport, cytochrome c to oxygen
    evidence_type: NAS
    original_reference_id: PMID:30030519
    review:
      summary: >-
        PMID:30030519 (Zong et al. 2018) reports the cryo-EM structure of the
        intact 14-subunit human Complex IV, which catalyzes electron transfer
        from cytochrome c to oxygen. COX4I1 is resolved as a structural subunit.
        NAS (Non-traceable Author Statement) from ComplexPortal.
      action: ACCEPT
      reason: >-
        Correct annotation. The reference describes the complete structure of
        the complex that performs this process, and COX4I1 is an integral subunit.
        Redundant with IBA annotation but correctly attributed.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: >-
            CIV is the terminal oxidase of the electron transport chain in
            mitochondria...It accepts electrons from cytochrome c to reduce the
            oxygen to water
  - term:
      id: GO:0031966
      label: mitochondrial membrane
    evidence_type: IDA
    original_reference_id: PMID:30030519
    review:
      summary: >-
        PMID:30030519 (Zong et al. 2018) provides cryo-EM structural evidence
        for COX4I1 in the mitochondrial inner membrane as part of Complex IV.
        ComplexPortal annotated this as IDA from the structural data.
      action: ACCEPT
      reason: >-
        Correct localization, directly demonstrated by cryo-EM structure.
        The more specific term "mitochondrial inner membrane" (GO:0005743)
        would be preferable but this broader annotation from the structural
        study is acceptable.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: >-
            we obtained the entire CIV structure containing 14 subunits
  - term:
      id: GO:0045333
      label: cellular respiration
    evidence_type: NAS
    original_reference_id: PMID:30030519
    review:
      summary: >-
        COX4I1 as a subunit of Complex IV participates in cellular respiration.
        Complex IV is the terminal enzyme of the respiratory chain, and
        cellular respiration encompasses the full OXPHOS pathway. This is a
        broad but correct process annotation from ComplexPortal.
      action: ACCEPT
      reason: >-
        Correct biological process annotation. Complex IV is an essential
        component of cellular respiration. However, more specific terms
        (GO:0006123 mitochondrial electron transport, cytochrome c to oxygen)
        are also annotated and are more informative. This broader term is
        acceptable as a parent-level annotation.
      supported_by:
        - reference_id: PMID:30030519
          supporting_text: Structure of the intact 14-subunit human cytochrome c oxidase.
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: HTP
    original_reference_id: PMID:34800366
    review:
      summary: >-
        PMID:34800366 (Morgenstern et al. 2021) is a quantitative high-confidence
        human mitochondrial proteome study. COX4I1 was identified as a high-
        confidence mitochondrial protein. HTP evidence from proteomics.
      action: ACCEPT
      reason: >-
        High-throughput proteomics confirming mitochondrial localization.
        Consistent with all other evidence.
      supported_by:
        - reference_id: PMID:34800366
          supporting_text: >-
            Quantitative high-confidence human mitochondrial proteome and its
            dynamics in cellular context
  - term:
      id: GO:0045277
      label: respiratory chain complex IV
    evidence_type: IMP
    original_reference_id: PMID:19393246
    review:
      summary: >-
        PMID:19393246 (Oswald et al. 2009) investigates COX17 knockdown effects
        on CIV assembly. The study shows that COX17 knockdown affects CIV
        assembly and supramolecular organization, and COX4I1 was observed in
        assembly intermediates. The IMP annotation indicates COX4I1 is part of
        CIV based on mutant phenotype data.
      action: ACCEPT
      reason: >-
        IMP evidence supporting COX4I1 as a component of Complex IV.
        The study demonstrates that perturbation of CIV assembly (via COX17
        knockdown) affects COX4I1-containing complexes, supporting its
        membership in CIV.
      supported_by:
        - reference_id: PMID:19393246
          supporting_text: >-
            COX17 siRNA knockdown affects activity and assembly of cytochrome c
            oxidase
  - term:
      id: GO:0005758
      label: mitochondrial intermembrane space
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9865412
    review:
      summary: >-
        Reactome pathway R-HSA-9865412 describes TIMM21-mediated transport of
        COX4, COX5A, COX6C to the MT-CO1:MITRAC complex. The IMS annotation
        reflects the C-terminal domain of COX4I1 that extends into the IMS.
      action: ACCEPT
      reason: >-
        TAS from Reactome. COX4I1 has an IMS-facing C-terminal domain
        (residues 125-169) as confirmed by UniProt topology annotations and
        cryo-EM structure. The annotation is technically correct.
      supported_by:
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            TOPO_DOM 125..169 /note="Mitochondrial intermembrane"
  - term:
      id: GO:0006119
      label: oxidative phosphorylation
    evidence_type: IEA
    original_reference_id: GO_REF:0000041
    review:
      summary: >-
        IEA annotation based on UniPathway vocabulary mapping (UPA00705).
        COX4I1 as a subunit of Complex IV directly participates in oxidative
        phosphorylation.
      action: ACCEPT
      reason: >-
        Correct process annotation. Complex IV is part of the OXPHOS system.
        UniProt explicitly states "PATHWAY: Energy metabolism; oxidative
        phosphorylation." This IEA correctly captures that COX4I1 participates
        in oxidative phosphorylation.
      supported_by:
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            PATHWAY: Energy metabolism; oxidative phosphorylation
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IDA
    original_reference_id: PMID:26746385
    review:
      summary: >-
        PMID:26746385 (Iwao et al. 2016) is about choline transporter-like
        proteins in human brain microvascular endothelial cells. The paper
        found CTL1 and CTL2 expressed on plasma membrane and mitochondria.
        COX4I1 was likely used as a mitochondrial marker in this study, not
        the primary subject.
      action: ACCEPT
      reason: >-
        IDA evidence for mitochondrial localization. Even though COX4I1 was
        likely used as a marker protein in this study, the localization result
        is correct and consistent with all other evidence.
      supported_by:
        - reference_id: PMID:26746385
          supporting_text: Functional expression of choline transporter like-protein
            1 (CTL1) and CTL2 in human brain microvascular endothelial cells.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: ISS
    original_reference_id: GO_REF:0000024
    review:
      summary: >-
        ISS annotation based on manual transfer from mouse ortholog P19783.
        COX4I1 localizes to the inner mitochondrial membrane as confirmed by
        direct structural evidence in human.
      action: ACCEPT
      reason: >-
        Correct localization. The ISS transfer from mouse is fully supported
        by direct human structural data (PMID:30030519) and UniProt annotation.
      supported_by:
        - reference_id: file:human/COX4I1/COX4I1-uniprot.txt
          supporting_text: >-
            SUBCELLULAR LOCATION: Mitochondrion inner membrane...Single-pass
            membrane protein
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IDA
    original_reference_id: PMID:15565177
    review:
      summary: >-
        PMID:15565177 (Stanelle et al. 2005) describes DIP (KIAA0767), a novel
        mitochondrial protein. COX4I1 was likely used as a mitochondrial marker
        in localization studies.
      action: ACCEPT
      reason: >-
        IDA evidence for mitochondrial localization, regardless of whether
        COX4I1 was a marker or target in this study. The localization is correct.
      supported_by:
        - reference_id: PMID:15565177
          supporting_text: A novel mitochondrial protein DIP mediates E2F1-induced
            apoptosis independently of p53.
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:26759378
    review:
      summary: >-
        PMID:26759378 (Cesnekova et al. 2016) demonstrates that LACE1/AFG1L
        (Q8WV93), a mitochondrial AAA-ATPase, physically interacts with COX4I1
        and mediates its degradation. This is a targeted, specific study showing
        that "LACE1...interacts physically with COX4 and COX5A subunits of
        complex IV." However, the annotation as generic "protein binding"
        does not capture the biological context of this interaction.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        While the LACE1-COX4I1 interaction is biologically meaningful (LACE1
        mediates degradation of nuclear-encoded CIV subunits including COX4),
        annotating it as generic "protein binding" is uninformative. This
        interaction is part of a quality control pathway for mitochondrial
        protein homeostasis, not a core function of COX4I1 itself.
      supported_by:
        - reference_id: PMID:26759378
          supporting_text: >-
            LACE1 mediates degradation of nuclear-encoded complex IV subunits
            COX4 (cytochrome c oxidase 4), COX5A and COX6A...the protein
            interacts physically with COX4 and COX5A subunits of complex IV and
            with mitochondrial inner-membrane protease YME1L
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: IPI
    original_reference_id: PMID:26321642
    review:
      summary: >-
        PMID:26321642 (Dennerlein et al. 2015) identifies MITRAC7 as a
        COX1-specific chaperone. COX4I1 was found in MITRAC complex
        intermediates along with MT-CO1 (P00395), SURF1 (Q15526), C12orf62
        (Q8N5G0), and COX14 (Q9Y2R0). This represents a genuine CIV assembly
        intermediate interaction.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Generic "protein binding" is uninformative. The interactions described
        are subunit-subunit contacts within CIV assembly intermediates, which
        are better captured by the part_of GO:0045277 and involvement in CIV
        assembly process annotations.
      supported_by:
        - reference_id: PMID:26321642
          supporting_text: >-
            The MITRAC complex represents the central assembly intermediate
            during this process as it receives imported subunits and regulates
            mitochondrial translation of COX1 mRNA

# ============================================================
# CC: Additional localization annotations
# ============================================================
  - term:
      id: GO:0016020
      label: membrane
    evidence_type: HDA
    original_reference_id: PMID:19946888
    review:
      summary: >-
        PMID:19946888 (Ghosh et al. 2010) defines the membrane proteome of NK
        cells using mass spectrometry. COX4I1 was identified in membrane
        fractions. The annotation to the very broad term "membrane" is correct
        but provides minimal information.
      action: ACCEPT
      reason: >-
        Correct but very general. COX4I1 is a membrane protein (inner
        mitochondrial membrane). The broad term "membrane" adds little
        information beyond what is already captured by the more specific
        inner membrane annotations, but it is not wrong.
      supported_by:
        - reference_id: PMID:19946888
          supporting_text: Defining the membrane proteome of NK cells.
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: HDA
    original_reference_id: PMID:20833797
    review:
      summary: >-
        PMID:20833797 (Zhao et al. 2011) is a phosphoproteomics study of
        functional mitochondria isolated from resting human muscle. COX4I1
        was identified in the mitochondrial phosphoproteome.
      action: ACCEPT
      reason: >-
        HDA evidence from mitochondrial proteomics confirming localization.
        Consistent with all other evidence. The study also identified
        phosphorylation sites in OXPHOS complexes, relevant to COX4I1's
        regulatory phosphorylation.
      supported_by:
        - reference_id: PMID:20833797
          supporting_text: >-
            Phosphoproteome analysis of functional mitochondria isolated from
            resting human muscle reveals extensive phosphorylation of inner
            membrane protein complexes and enzymes

# ============================================================
# CC: Reactome TAS inner membrane annotations
# ============================================================
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-163214
    review:
      summary: >-
        Reactome pathway R-HSA-163214 (Electron transfer from reduced cytochrome
        c to molecular oxygen) places COX4I1 in the mitochondrial inner membrane
        as part of CIV.
      action: ACCEPT
      reason: >-
        Correct TAS annotation from Reactome. COX4I1 is in the inner
        mitochondrial membrane as part of Complex IV, which catalyzes electron
        transfer from cytochrome c to oxygen.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9709406
    review:
      summary: >-
        Reactome pathway R-HSA-9709406 (CO binds to Cytochrome c oxidase)
        places COX4I1 in the mitochondrial inner membrane. This pathway
        describes carbon monoxide binding to CIV.
      action: ACCEPT
      reason: >-
        Correct TAS localization. COX4I1 is in the inner mitochondrial
        membrane as a CIV subunit.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9865412
    review:
      summary: >-
        Reactome pathway R-HSA-9865412 (TIMM21 carries COX4, COX5A, COX6C to
        MT-CO1:MITRAC) places COX4I1 in the inner membrane during CIV assembly.
      action: ACCEPT
      reason: >-
        Correct TAS localization from CIV assembly pathway in Reactome.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9865449
    review:
      summary: >-
        Reactome pathway R-HSA-9865449 (Metallochaperone inserts Cu2+ into
        MT-CO1) places COX4I1 in the inner membrane during copper insertion
        step of CIV assembly.
      action: ACCEPT
      reason: >-
        Correct TAS localization from CIV assembly pathway in Reactome.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9865579
    review:
      summary: >-
        Reactome pathway R-HSA-9865579 (MT-CO1 and MT-CO2 complexes associate,
        installing heme moieties) places COX4I1 in the inner membrane during
        CIV assembly.
      action: ACCEPT
      reason: >-
        Correct TAS localization from CIV assembly pathway in Reactome.
  - term:
      id: GO:0005743
      label: mitochondrial inner membrane
    evidence_type: TAS
    original_reference_id: Reactome:R-HSA-9865663
    review:
      summary: >-
        Reactome pathway R-HSA-9865663 (MT-CO3, COX6A,B,7A and NDUFA4 bind to
        holo-MT-CO1,2 complex) places COX4I1 in the inner membrane during late
        CIV assembly.
      action: ACCEPT
      reason: >-
        Correct TAS localization from CIV assembly pathway in Reactome.

# ============================================================
# MF: cytochrome-c oxidase activity
# ============================================================
  - term:
      id: GO:0004129
      label: cytochrome-c oxidase activity
    evidence_type: TAS
    original_reference_id: PMID:2157630
    review:
      summary: >-
        PMID:2157630 (Lomax et al. 1990) describes chromosomal mapping of human
        COX4 using a chimpanzee pseudogene. The paper identifies COX4I1 as a
        subunit of cytochrome c oxidase (EC 1.9.3.1) and maps the gene to
        chromosome 16. This is a TAS (Traceable Author Statement) annotation
        from the early characterization of the gene. For complex subunits,
        the qualifier should be "contributes_to" rather than "enables" since
        COX4I1 is a non-catalytic regulatory subunit.
      action: ACCEPT
      reason: >-
        COX4I1 is a structural and regulatory subunit of CIV, which has
        cytochrome-c oxidase activity. The appropriate GO annotation pattern
        for non-catalytic complex subunits is "contributes_to" for complex-level
        molecular function. COX4I1 contributes to CIV activity through
        structural support, assembly, and allosteric regulation, though it does
        not itself catalyze the electron transfer reaction. The GOA file shows
        the qualifier as "enables" but this should ideally be "contributes_to"
        per GO annotation conventions for complex subunits.
      supported_by:
        - reference_id: PMID:2157630
          supporting_text: >-
            subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1)
        - reference_id: file:human/COX4I1/COX4I1-deep-research-falcon.md
          supporting_text: >-
            COX4-1 integrates matrix adenine nucleotide/PKA signals to adjust
            complex IV turnover to cellular energy state, supports cytochrome c
            docking via COX2 interactions, and participates in assembly and
            supercomplex formation

# ============================================================
# BP: generation of precursor metabolites and energy
# ============================================================
  - term:
      id: GO:0006091
      label: generation of precursor metabolites and energy
    evidence_type: TAS
    original_reference_id: PMID:2157630
    review:
      summary: >-
        Very broad biological process annotation. COX4I1 as part of Complex IV
        contributes to generation of precursor metabolites and energy through
        oxidative phosphorylation. However, this is an extremely general term
        that adds little specificity beyond the more precise OXPHOS and electron
        transport annotations already present.
      action: ACCEPT
      reason: >-
        Correct but very broad. More specific process annotations (GO:0006123,
        GO:0006119, GO:0045333) are also present and are more informative.
        This annotation is technically correct as CIV drives proton pumping
        that powers ATP synthesis.

# ============================================================
# NEW annotations: suggested additions
# ============================================================
      supported_by:
        - reference_id: PMID:2157630
          supporting_text: Novel use of a chimpanzee pseudogene for chromosomal mapping
            of human cytochrome c oxidase subunit IV.
  - term:
      id: GO:0005524
      label: ATP binding
    evidence_type: IDA
    original_reference_id: PMID:21641552
    review:
      summary: >-
        COX4I1 has a well-characterized ATP/ADP-binding allosteric site in its
        matrix-facing domain. ATP binding at high ATP/ADP ratios inhibits
        Complex IV activity. This has been demonstrated experimentally, notably
        by Acin-Perez et al. (2011, PMID:21641552) who showed that
        phosphorylation of Ser58 by PKA prevents ATP binding and relieves
        inhibition. The deep research review confirms "An ATP-binding site in
        COX4-1's matrix domain mediates allosteric inhibition of complex IV
        when ATP/ADP is high."
      action: NEW
      reason: >-
        ATP binding is a specific molecular function of COX4I1 that is not
        represented in the current GO annotation set. This is the basis of
        COX4I1's key regulatory function - allosteric inhibition of Complex IV
        at high ATP/ADP ratios. This is a subunit-specific MF annotation
        distinct from the complex-level cytochrome-c oxidase activity.
      additional_reference_ids: ["PMID:21641552"]
      supported_by:
        - reference_id: PMID:21641552
          supporting_text: >-
            Protein phosphorylation and prevention of cytochrome oxidase inhibition
            by ATP: coupled mechanisms of energy metabolism regulation.
        - reference_id: file:human/COX4I1/COX4I1-deep-research-falcon.md
          supporting_text: >-
            An ATP-binding site in COX4-1's matrix domain mediates allosteric
            inhibition of complex IV when ATP/ADP is high; phosphorylation of
            Ser58 by intramitochondrial PKA (sAC-cAMP-PKA axis) prevents ATP
            binding and relieves inhibition, functioning as a rapid bioenergetic
            switch
  - term:
      id: GO:1904959
      label: regulation of cytochrome-c oxidase activity
    evidence_type: IDA
    original_reference_id: PMID:21641552
    review:
      summary: >-
        COX4I1 is the primary regulatory subunit of Complex IV, mediating
        allosteric feedback inhibition through its ATP/ADP binding site and
        through PKA-dependent phosphorylation at Ser58. This regulatory
        function is the defining characteristic that distinguishes COX4I1
        from purely structural CIV subunits. "Regulation of cytochrome-c
        oxidase activity" (GO:1904959) precisely captures this core function.
      action: NEW
      reason: >-
        This is a key missing annotation. COX4I1's regulatory function through
        allosteric ATP binding and PKA phosphorylation is its primary
        distinguishing role. The deep research review emphasizes this:
        "noncatalytic, COX4-1 regulates complex IV turnover and assembly...
        integrates metabolic signals via adenine nucleotide binding."
      additional_reference_ids: ["PMID:21641552"]
      supported_by:
        - reference_id: PMID:21641552
          supporting_text: >-
            Protein phosphorylation and prevention of cytochrome oxidase inhibition
            by ATP: coupled mechanisms of energy metabolism regulation.
        - reference_id: file:human/COX4I1/COX4I1-deep-research-falcon.md
          supporting_text: >-
            COX4-1 modulates this pacemaker step of the electron transport chain
            through allosteric control...phosphorylation of Ser58 by
            intramitochondrial PKA...prevents ATP binding and relieves inhibition
  - term:
      id: GO:0033617
      label: mitochondrial respiratory chain complex IV assembly
    evidence_type: TAS
    original_reference_id: PMID:23260140
    review:
      summary: >-
        COX4I1 participates in early CIV assembly intermediates (MITRAC
        complexes). It is one of the first nuclear-encoded subunits to be
        incorporated during assembly (COX4-COX5A module). Multiple publications
        demonstrate COX4I1's role in CIV assembly, including PMID:23260140
        (MITRAC complexes) and PMID:26321642 (MITRAC7 assembly checkpoint).
        This process is not currently annotated for COX4I1.
      action: NEW
      reason: >-
        COX4I1 is a key participant in CIV assembly. The deep research review
        states "COX4 participates in early assembly modules (COX4-COX5A)."
        Multiple Reactome pathways describe COX4I1's role in assembly steps.
        This is a core biological process annotation that is missing from the
        current set.
      additional_reference_ids: ["PMID:23260140", "PMID:26321642"]
      supported_by:
        - reference_id: PMID:23260140
          supporting_text: >-
            we report a comprehensive dissection of early cytochrome c oxidase
            assembly intermediates
        - reference_id: file:human/COX4I1/COX4I1-deep-research-falcon.md
          supporting_text: >-
            COX4 participates in early assembly modules (COX4-COX5A) and
            contributes to respirasome (CI-CIII2-CIV) formation
core_functions:
  - molecular_function:
      id: GO:0005524
      label: ATP binding
    contributes_to_molecular_function:
      id: GO:0004129
      label: cytochrome-c oxidase activity
    directly_involved_in:
      - id: GO:1904959
        label: regulation of cytochrome-c oxidase activity
      - id: GO:0006123
        label: mitochondrial electron transport, cytochrome c to oxygen
      - id: GO:0033617
        label: mitochondrial respiratory chain complex IV assembly
    locations:
      - id: GO:0005743
        label: mitochondrial inner membrane
    description: >-
      COX4I1 is the principal regulatory supernumerary subunit of cytochrome c
      oxidase (Complex IV). It binds ATP at a matrix-exposed allosteric site and
      integrates adenine nucleotide and cAMP/PKA signaling to modulate Complex IV
      catalytic output. As a non-catalytic subunit, COX4I1 contributes_to
      cytochrome-c oxidase activity while directly participating in regulation of
      that activity, mitochondrial electron transport from cytochrome c to oxygen,
      and early Complex IV assembly (MITRAC-associated steps).
    supported_by:
      - reference_id: PMID:21641552
        supporting_text: >-
          Protein phosphorylation and prevention of cytochrome oxidase inhibition
          by ATP:
          coupled mechanisms of energy metabolism regulation.
      - reference_id: PMID:30030519
        supporting_text: >-
          we obtained the entire CIV structure containing 14 subunits, which
          includes the extra subunit NDUFA4
      - reference_id: file:human/COX4I1/COX4I1-deep-research-falcon.md
        supporting_text: >-
          COX4 participates in early assembly modules (COX4-COX5A) and
          contributes to respirasome (CI-CIII2-CIV) formation
    in_complex:
      id: GO:0045277
      label: respiratory chain complex IV
references:
  - id: GO_REF:0000002
    title: Gene Ontology annotation through association of InterPro records with GO
      terms
    findings:
      - statement: InterPro2GO mapping (COX4 family, IPR004203/IPR036639) propagates
          Complex IV-related electron transport annotations to COX4I1.
  - id: GO_REF:0000024
    title: Manual transfer of experimentally-verified manual GO annotation data to
      orthologs by curator judgment of sequence similarity
    findings:
      - statement: ISS curator transfer from mouse COX4I1 (P19783) propagates inner
          mitochondrial membrane localization to human COX4I1.
  - id: GO_REF:0000033
    title: Annotation inferences using phylogenetic trees
    findings:
      - statement: PAINT/IBA phylogenetic propagation supports Complex IV membership
          and cytochrome-c-to-O2 electron transport for COX4I1 across COX4 family
          orthologs.
  - id: GO_REF:0000041
    title: Gene Ontology annotation based on UniPathway vocabulary mapping
    findings:
      - statement: UniPathway UPA00705 (oxidative phosphorylation) propagates participation
          in the OXPHOS pathway to COX4I1.
  - id: GO_REF:0000052
    title: Gene Ontology annotation based on curation of immunofluorescence data
    findings:
      - statement: Curated immunofluorescence data (Human Protein Atlas) place COX4I1
          in mitochondria.
  - id: GO_REF:0000107
    title: Automatic transfer of experimentally verified manual GO annotation data
      to orthologs using Ensembl Compara
    findings:
      - statement: Ensembl Compara automatic ortholog transfer (from rodent COX4I1)
          propagates mitochondrial localization and energy-status response annotations
          to human COX4I1.
  - id: GO_REF:0000108
    title: Automatic assignment of GO terms using logical inference, based on on inter-ontology
      links
    findings:
      - statement: Logical inference from cytochrome-c oxidase activity (GO:0004129)
          propagates proton transmembrane transport (GO:1902600) to Complex IV
          subunits including COX4I1.
  - id: GO_REF:0000117
    title: Electronic Gene Ontology annotations created by ARBA machine learning models
    findings:
      - statement: ARBA machine-learning rules assign intermembrane-space topology
          to COX4I1 consistent with its IMS-facing C-terminal segment.
  - id: GO_REF:0000120
    title: Combined Automated Annotation using Multiple IEA Methods
    findings:
      - statement: Combined automated IEA methods propagate inner mitochondrial
          membrane localization and Complex IV membership to COX4I1.
  - id: PMID:2157630
    title: Novel use of a chimpanzee pseudogene for chromosomal mapping of human cytochrome
      c oxidase subunit IV.
    findings:
      - statement: Identifies COX4I1 as subunit IV of cytochrome c oxidase (EC 1.9.3.1)
          and maps the gene to chromosome 16q22-qter.
        supporting_text: Novel use of a chimpanzee pseudogene for chromosomal mapping
          of human cytochrome c oxidase subunit IV.
  - id: PMID:15565177
    title: A novel mitochondrial protein DIP mediates E2F1-induced apoptosis independently
      of p53.
    findings:
      - statement: COX4I1 was likely used as a mitochondrial marker; the study focuses
          on the novel protein DIP.
        supporting_text: A novel mitochondrial protein DIP mediates E2F1-induced apoptosis
          independently of p53.
  - id: PMID:19393246
    title: Knockdown of human COX17 affects assembly and supramolecular organization
      of cytochrome c oxidase.
    findings:
      - statement: COX17 knockdown affects CIV assembly and supercomplex organization;
          COX4I1 observed in assembly intermediates.
        supporting_text: Knockdown of human COX17 affects assembly and supramolecular
          organization of cytochrome c oxidase.
  - id: PMID:19946888
    title: Defining the membrane proteome of NK cells.
    findings:
      - statement: Proteomic identification of COX4I1 in NK cell membrane fractions.
        supporting_text: Defining the membrane proteome of NK cells.
  - id: PMID:20833797
    title: Phosphoproteome analysis of functional mitochondria isolated from resting
      human muscle reveals extensive phosphorylation of inner membrane protein complexes
      and enzymes.
    findings:
      - statement: Identified COX4I1 in the mitochondrial phosphoproteome from human
          skeletal muscle.
        supporting_text: Phosphoproteome analysis of functional mitochondria isolated
          from resting human muscle reveals extensive phosphorylation of inner membrane
          protein complexes and enzymes.
  - id: PMID:21641552
    title: 'Protein phosphorylation and prevention of cytochrome oxidase inhibition
      by ATP: coupled mechanisms of energy metabolism regulation.'
    findings:
      - statement: >-
          Demonstrated that PKA-dependent phosphorylation of Ser58 on COX4-1 prevents
          allosteric ATP inhibition of Complex IV, coupling cAMP signaling to
          mitochondrial respiration regulation.
        supporting_text: >-
          Protein phosphorylation and prevention of cytochrome oxidase inhibition
          by ATP:
          coupled mechanisms of energy metabolism regulation.
  - id: PMID:23260140
    title: MITRAC links mitochondrial protein translocation to respiratory-chain assembly
      and translational regulation.
    findings:
      - statement: >-
          Identified COX4I1 in MITRAC (mitochondrial translation regulation assembly
          intermediate of cytochrome c oxidase) complexes, linking protein import
          to
          CIV assembly.
        supporting_text: MITRAC links mitochondrial protein translocation to respiratory-chain
          assembly and translational regulation.
  - id: PMID:25416956
    title: A proteome-scale map of the human interactome network.
    findings:
      - statement: Large-scale Y2H interactome; COX4I1 interaction with SDCBP reported.
        supporting_text: A proteome-scale map of the human interactome network.
  - id: PMID:26321642
    title: MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint during
      Cytochrome c Oxidase Assembly.
    findings:
      - statement: >-
          COX4I1 found in MITRAC complexes during CIV assembly; MITRAC7 acts as
          COX1-specific chaperone in the assembly pathway.
        supporting_text: MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint
          during Cytochrome c Oxidase Assembly.
  - id: PMID:26746385
    title: Functional expression of choline transporter like-protein 1 (CTL1) and
      CTL2 in human brain microvascular endothelial cells.
    findings:
      - statement: COX4I1 used as mitochondrial marker; mitochondrial localization
          confirmed.
        supporting_text: Functional expression of choline transporter like-protein
          1 (CTL1) and CTL2 in human brain microvascular endothelial cells.
  - id: PMID:26759378
    title: The mammalian homologue of yeast Afg1 ATPase (lactation elevated 1) mediates
      degradation of nuclear-encoded complex IV subunits.
    findings:
      - statement: >-
          LACE1/AFG1L physically interacts with COX4I1 and COX5A and mediates their
          degradation as part of mitochondrial protein quality control.
        supporting_text: The mammalian homologue of yeast Afg1 ATPase (lactation elevated
          1) mediates degradation of nuclear-encoded complex IV subunits.
  - id: PMID:28514442
    title: Architecture of the human interactome defines protein communities and disease
      networks.
    findings:
      - statement: BioPlex 2.0 AP-MS interactome; COX4I1 co-purification with DBT
          reported.
        supporting_text: Architecture of the human interactome defines protein communities
          and disease networks.
  - id: PMID:28766551
    title: Mutation in the COX4I1 gene is associated with short stature, poor weight
      gain and increased chromosomal breaks, simulating Fanconi anemia.
    findings:
      - statement: >-
          Homozygous COX4I1 K101N variant causes Complex IV deficiency with growth
          failure and chromosomal instability. Functional rescue with wild-type
          COX4I1 restored COX activity.
        supporting_text: Mutation in the COX4I1 gene is associated with short stature,
          poor weight gain and increased chromosomal breaks, simulating Fanconi anemia.
  - id: PMID:30030519
    title: Structure of the intact 14-subunit human cytochrome c oxidase.
    findings:
      - statement: >-
          Cryo-EM structure (3.3A) of human CIV as part of supercomplex,
          confirming 14-subunit monomer composition including COX4I1. COX4I1
          resolved with transmembrane helix, matrix domain, and IMS domain.
        supporting_text: Structure of the intact 14-subunit human cytochrome c oxidase.
  - id: PMID:31515488
    title: Extensive disruption of protein interactions by genetic variants across
      the allele frequency spectrum in human populations.
    findings:
      - statement: Study of how genetic variants affect protein interactions; COX4I1-SDCBP
          interaction tested.
        supporting_text: Extensive disruption of protein interactions by genetic variants
          across the allele frequency spectrum in human populations.
  - id: PMID:32814053
    title: Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins
      and Uncovers Widespread Protein Aggregation in Affected Brains.
    findings:
      - statement: Neurodegenerative disease interactome mapping; COX4I1-KLF11 interaction
          reported.
        supporting_text: Interactome Mapping Provides a Network of Neurodegenerative
          Disease Proteins and Uncovers Widespread Protein Aggregation in Affected
          Brains.
  - id: PMID:33961781
    title: Dual proteome-scale networks reveal cell-specific remodeling of the human
      interactome.
    findings:
      - statement: >-
          BioPlex 3.0 AP-MS; COX4I1 co-purification with MT-CO1 (genuine CIV
          subunit interaction) and DBT reported.
        supporting_text: Dual proteome-scale networks reveal cell-specific remodeling
          of the human interactome.
  - id: PMID:34800366
    title: Quantitative high-confidence human mitochondrial proteome and its dynamics
      in cellular context.
    findings:
      - statement: High-confidence mitochondrial proteome identifies COX4I1 as a mitochondrial
          protein.
        supporting_text: Quantitative high-confidence human mitochondrial proteome
          and its dynamics in cellular context.
  - id: Reactome:R-HSA-163214
    title: Electron transfer from reduced cytochrome c to molecular oxygen
    findings:
      - statement: COX4I1 participates in Complex IV-mediated electron transfer.
        supporting_text: Electron transfer from reduced cytochrome c to molecular
          oxygen
  - id: Reactome:R-HSA-9709406
    title: CO binds to Cytochrome c oxidase
    findings:
      - statement: COX4I1 is a subunit of CIV, the target of CO binding.
        supporting_text: CO binds to Cytochrome c oxidase
  - id: Reactome:R-HSA-9865412
    title: TIMM21 carries COX4, COX5A, COX6C to MT-CO1:MITRAC
    findings:
      - statement: COX4I1 is transported by TIMM21 to MT-CO1:MITRAC assembly intermediate.
        supporting_text: TIMM21 carries COX4, COX5A, COX6C to MT-CO1:MITRAC
  - id: Reactome:R-HSA-9865449
    title: Metallochaperone inserts Cu2+ into MT-CO1
    findings:
      - statement: COX4I1 is present during copper insertion step of CIV assembly.
        supporting_text: Metallochaperone inserts Cu2+ into MT-CO1
  - id: Reactome:R-HSA-9865579
    title: MT-CO1 and MT-CO2 complexes associate, installing heme moieties
    findings:
      - statement: COX4I1 present during MT-CO1/MT-CO2 module joining in CIV assembly.
        supporting_text: MT-CO1 and MT-CO2 complexes associate, installing heme moieties
  - id: Reactome:R-HSA-9865663
    title: MT-CO3, COX6A,B,7A and NDUFA4 bind to holo-MT-CO1,2 complex
    findings:
      - statement: COX4I1 already incorporated during late CIV assembly steps.
        supporting_text: MT-CO3, COX6A,B,7A and NDUFA4 bind to holo-MT-CO1,2 complex
suggested_questions:
  - question: >-
      What is the structural and energetic basis of allosteric ATP/ADP binding at the
      matrix-facing site of COX4-1, and how does Ser58 phosphorylation by intramitochondrial
      PKA propagate to alter cytochrome-c oxidase turnover? A residue-level mechanism would
      sharpen the molecular-function annotation for COX4I1 beyond the current
      "regulation of cytochrome-c oxidase activity" term.
  - question: >-
      Under physiological conditions in human tissues, what is the relative contribution
      of COX4-1 (vs. COX4-2) to respirasome (CI-CIII2-CIV) assembly and to oxygen-affinity
      tuning, and which signals (HIF, redox, hormone) drive isoform switching in vivo?
      This would clarify whether respirasome assembly and isoform-dependent O2 kinetics
      warrant additional, more specific BP annotations.
  - question: >-
      Beyond the K101N and P152T variants, is the chromosomal-instability phenotype of
      MC4DN16 a direct consequence of bioenergetic failure (ATP/ROS imbalance) or does
      COX4I1 have a moonlighting role in genome stability? Clarifying this would
      determine whether genome-stability process annotations are warranted.
suggested_experiments:
  - description: >-
      Cryo-EM of intact human Complex IV reconstituted with COX4I1 carrying alanine
      substitutions at the allosteric ATP-binding pocket (matrix domain) and at Ser58,
      compared with phospho-mimetic S58D and wild-type, in the presence of saturating
      ATP/ADP. Quantify activity, ATP-binding affinity, and global conformational
      changes by HDX-MS.
    hypothesis: >-
      The ATP/ADP allosteric site directly transmits a conformational signal to the
      cytochrome c docking face, and Ser58 phosphorylation eliminates ATP binding by
      occluding the pocket rather than indirectly altering domain dynamics.
    experiment_type: structural biology / biochemistry
  - description: >-
      Isoform-swap CRISPR knock-in in matched human cell lines (e.g., HEK293, lung A549,
      brain SH-SY5Y) replacing endogenous COX4I1 with COX4I2 or vice versa, followed by
      high-resolution respirometry, blue native PAGE supercomplex profiling, and ROS
      measurements across a physiological O2 range (1-21% O2). Compare with HIF-1α
      stabilization (DMOG) to dissect transcriptional vs. compositional effects.
    hypothesis: >-
      COX4-1 to COX4-2 isoform exchange is sufficient to raise CIV p50 (lower O2
      affinity) and remodel supercomplex distribution in human cells, independently of
      broader HIF-1 transcriptional programs.
    experiment_type: cell biology / respirometry
  - description: >-
      Patient-derived fibroblast and iPSC-cardiomyocyte models of COX4I1 K101N and P152T
      complemented with wild-type or phospho-dead (S58A) COX4I1, with quantitative
      analysis of CIV holocomplex assembly (BN-PAGE), supercomplex formation, ATP
      production, ROS, and chromosomal breakage after diepoxybutane challenge.
    hypothesis: >-
      Genome instability in MC4DN16 patient cells is rescued by restoring CIV bioenergetic
      output, supporting an indirect (ROS/energy-mediated) rather than a direct moonlighting
      role for COX4I1 in DNA-damage responses.
    experiment_type: clinical model / cell biology