cmc4 (systematic name SPAC4F10.22; synonym tam2) is a small (70-residue) mitochondrial intermembrane space (IMS) protein of the twin CX9C / coiled-coil- helix-coiled-coil-helix (CHCH) family, the sole fission-yeast member of the conserved CMC4 (Cx9C motif-containing protein 4) family (PANTHER PTHR15590, Pfam PF08991, InterPro IPR027179). Its two CX9C motifs form a helical hairpin stabilized by two intramolecular disulfide bonds; like other twin CX9C proteins it is imported into the IMS and oxidatively folded by the MIA40 (Mia40/CHCHD4)- Erv1 disulfide relay, which recognizes the cysteine motifs. CMC4 orthologs are broadly conserved from yeasts to human (human CMC4, P56277). The family is associated with cytochrome c oxidase (COX) biogenesis: in human cells CMC4 physically associates with the COX assembly factor SCO1, implicating it in COX II sub-assembly, although CMC4 is non-essential for respiration and no specific molecular activity has been demonstrated for it. In S. pombe the gene was identified as a meiotically regulated transcript (tam2), and its deletion is viable with no reported growth or respiratory phenotype.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005758 mitochondrial intermembrane space | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference of mitochondrial intermembrane space localization from the CMC4 family (PANTHER PTHR15590 / PTN000400520). This is well supported: the twin CX9C / CHCH fold and the MIA40-Erv1 import route are diagnostic of soluble IMS proteins, and the human/yeast orthologs are IMS-localized. This is the single substantive, defensible annotation for cmc4 and represents its core cellular context. Reason: Localization is strongly supported by family membership (twin CX9C, MIA40-import) and ortholog data; the IBA inference from PTHR15590 is appropriate. Note the localization has not been demonstrated experimentally in S. pombe itself. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000400520 Β· CMC4 family (PTHR15590) SUPPORTS TRANSFER Twin CX9C / MIA40-imported IMS protein family; human and yeast orthologs are IMS-localized. Supporting Evidence: PMID:33498264 CMC4 is a CX9C family member that localizes to the mitochondrial IMS |
| GO:0005758 mitochondrial intermembrane space | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation of the same localization derived from the UniProtKB/Swiss-Prot subcellular-location keyword (SL-0169). Redundant with, and consistent with, the IBA annotation above. Reason: Consistent with the family-supported IMS localization; the electronic SubCell mapping mirrors the UniProt annotation and does not overreach. |
| GO:0003674 molecular_function | ND GO_REF:0000015 | ACCEPT | Summary: Molecular function is recorded as ND (no data). No specific biochemical activity has been demonstrated for cmc4 or any CMC4 ortholog: unlike the related copper chaperones Cmc1/Cmc2, CMC4 has no demonstrated metal binding or catalytic activity, and its only functional lead is a physical association with SCO1 in human cells. The ND root placeholder honestly reflects a genuine molecular-function knowledge gap. Reason: No experimental or defensible computational molecular-function term can be asserted. Retaining the ND annotation correctly signals an undetermined molecular function rather than forcing an unsupported term. The gene's search record (notes) finds no study of cmc4 in S. pombe that identifies a molecular function. Supporting Evidence: file:SCHPO/cmc4/cmc4-notes.md No defined **molecular function** for cmc4/CMC4 Knowledge gap: The molecular activity of cmc4 is undetermined. It is unknown whether it acts as a metallochaperone, a redox/disulfide-relay accessory factor, or a protein-interaction adaptor within the mitochondrial IMS, and no direct binding partner has been identified in S. pombe. OPEN BIOLOGY MF_DARK Resolve: Affinity/proximity proteomics of tagged cmc4 in S. pombe to identify direct partners; metal-binding and thiol-redox assays on recombinant protein to test metallochaperone vs disulfide-relay-accessory models. "determined that CMC4 interacts with SCO1 in human cell cultures" β PMID:33498264 |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Biological process is recorded as ND (no data). A COX II sub-assembly role is only inferred from the human SCO1 interaction, is non-essential, and has not been tested in S. pombe; tam2Ξ is viable with no reported respiratory or meiotic phenotype despite the transcript being meiotically regulated. The ND placeholder honestly reflects an undetermined biological process. Reason: No experimentally supported biological process for cmc4 in S. pombe. The COX-assembly link is cross-species and non-essential; the meiotic transcript regulation (tam2) has no assigned meiotic function. ND is the correct, honest annotation. The gene's search record (notes) finds no study of cmc4 in S. pombe that identifies a biological process. Supporting Evidence: file:SCHPO/cmc4/cmc4-notes.md No demonstrated **biological process** for cmc4 in pombe: GOA has GO:0008150 ND. Knowledge gap: The in vivo biological role of cmc4 in S. pombe is unknown: whether it contributes to cytochrome c oxidase assembly/respiration (as suggested for the human ortholog) or has a distinct meiosis-related role implied by its tam2 expression pattern is undetermined, and no deletion phenotype has been reported. OPEN BIOLOGY BP_DARK Resolve: Phenotyping of cmc4Ξ (respiratory growth on non-fermentable carbon, COX activity/assembly; sporulation, spore viability and meiotic timing) and epistasis with cox11/sco1 orthologs. "While tam2 .Ξ was viable, new21 was not" β PMID:21270388 |
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Download this section (compressed HTML)Q: Does cmc4 physically associate with an S. pombe SCO1/Cox-assembly ortholog, as its human counterpart does, or with a distinct IMS partner?
Q: Is cmc4 required for respiratory growth or cytochrome c oxidase activity/assembly in fission yeast, or is it fully dispensable as the viability of tam2Ξ and the lack of a respiratory phenotype in yeast CMC4 knockdown suggest?
Q: Does the meiotic (tam2) regulation of cmc4 reflect a meiosis- or sporulation-specific function, or merely coordinate expression of a mitochondrial protein with increased respiratory demand during differentiation?
Q: Does cmc4 bind a metal ion (e.g. copper) or participate directly in the MIA40-Erv1 disulfide relay as a substrate/accessory factor?
Experiment: Proximity labeling (BioID/TurboID) or affinity purification of endogenously tagged cmc4 in S. pombe under fermentative and respiratory (glycerol/ethanol) growth to identify direct IMS partners and test for association with Sco1/COX assembly factors.
Hypothesis: cmc4 associates with a COX II copper-delivery assembly factor (Sco1 ortholog) in the IMS, conserving the human CMC4-SCO1 interaction.
Experiment: Phenotypic characterization of a cmc4Ξ strain: growth on non-fermentable carbon sources, spectrophotometric cytochrome c oxidase activity and BN-PAGE COX assembly, and meiotic assays (sporulation efficiency, spore viability, meiotic progression timing).
Hypothesis: cmc4 loss produces a subtle, condition-specific respiratory and/or meiotic phenotype consistent with a non-essential accessory role in COX assembly.
Experiment: Biochemical characterization of recombinant cmc4: metal-binding assays (particularly Cu(I)) and thiol-disulfide/redox assays to test whether cmc4 is a metallochaperone or a MIA40-Erv1 relay accessory factor.
Hypothesis: cmc4 either binds a transition-metal ion or engages the disulfide relay beyond its own oxidative folding, defining its molecular activity.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: cmc4 is a functionally dark gene: its molecular activity, its direct binding partner(s)/client in S. pombe, and its in vivo biological role are all undetermined. It is unresolved whether cmc4 acts in cytochrome c oxidase (COX II) assembly (as inferred from the human CMC4-SCO1 interaction) or has a distinct role connected to its meiotic (tam2) expression, and no cmc4Ξ phenotype has been reported.
OPEN BIOLOGY MF_DARK
What is known: What is firmly established: cmc4 (SPAC4F10.22 / tam2) is a small 70-residue mitochondrial intermembrane space protein of the twin CX9C / CHCH (CMC4) family, with two CX9C motifs forming a disulfide-bonded helical hairpin, imported and oxidatively folded by the MIA40-Erv1 disulfide relay. It is the sole fission-yeast member of a family conserved to human (CMC4, P56277). The human ortholog physically associates with the COX assembly factor SCO1, implicating the family in a non-essential branch of COX II sub-assembly, and the transcript is meiotically regulated in S. pombe (tam2), whose deletion is viable.
Significance: CMC4 is a conserved member of the twin CX9C protein family that collectively governs cytochrome c oxidase biogenesis and mitochondrial redox homeostasis, yet it is one of the least characterized members. Resolving its activity and in vivo role would close a genuine unknome gap and test whether the human COX II sub-assembly association is conserved and functionally important in a genetically tractable model.
What would resolve it: Affinity/proximity proteomics of tagged cmc4 in S. pombe to identify direct partners (test association with Sco1/Cox-assembly orthologs); recombinant metal-binding and thiol-disulfide assays to distinguish metallochaperone from disulfide-relay-accessory models; and phenotyping of cmc4Ξ for respiratory (COX activity on non-fermentable carbon) and meiotic (sporulation, spore viability, timing) defects.
Provenance (the field's own admissions):
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