UQCC6 (BRAWNIN, C12orf73) is a 71-amino-acid microprotein encoded by a small open reading frame and conserved across vertebrates. It is an integral protein of the mitochondrial inner membrane, anchored by a single N-terminal signal-anchor helix with its short N terminus in the matrix and its C-terminal region in the intermembrane space. UQCC6 is a non-catalytic assembly factor for respiratory chain complex III (ubiquinol-cytochrome c reductase, cytochrome bc1). Together with the microprotein UQCC5 (SMIM4) and the cytochrome b translation/assembly factors UQCC1, UQCC2 and UQCC4 (C16orf91) it forms an early assembly intermediate (termed the COMB complex) that binds newly synthesised mitochondrially encoded cytochrome b (MT-CYB) and stabilises it before later subunits join. Loss of the protein in zebrafish or mouse cells causes near-complete loss of assembled complex III dimers with little effect on the other respiratory complexes, leading in zebrafish to lactic acidosis, growth retardation and early death; partial knockdown in human cells has much milder effects. Its expression is highest in cardiac and skeletal muscle and is induced by nutrient stress and AMPK activation, linking complex III biogenesis to cellular energy demand.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:32161263 Mitochondrial peptide BRAWNIN is essential for vertebrate re... | REMOVE | Summary: Co-immunoprecipitation of BRAWNIN with the complex III core subunit UQCRC1 (UniProtKB:P31930), found by IP/MS in HEK293T mitochondria and validated by co-IP in mouse heart mitochondria. Reason: Bare protein binding carries no functional information, and no more informative molecular function can be supported from this evidence, so the row is removed rather than rewritten. The functionally relevant partners of UQCC6 are early assembly factors (UQCC1, UQCC2, UQCC5/SMIM4) and nascent cytochrome b; an independent study (PMID:34969438) did not find UQCC6 associated with late complex III constituents and recovered only marginal amounts with UQCRQ. The complex III assembly role is captured by the GO:0034551 process annotations. Removal does not imply that the reported interaction is false. Supporting Evidence: PMID:32161263 In contrast, the interaction between BR and UQCRC1 detected in human cells was validated by co-immunoprecipitation(co-IP)/western blot in digitonin-solubilized mouse heart mitochondria expressing a Br-FLAG construct PMID:34969438 In contrast to the reports on BR (Zhang et al., 2020), C12ORF73 does not interact with late complex constituents |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput assignment to the high-confidence human mitochondrial proteome (MitoCoP). Reason: Correct, consistent with all targeted localisation studies; less specific than the mitochondrial inner membrane annotations also present. Supporting Evidence: PMID:34800366 We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that BRAWNIN family members act in mitochondria. Reason: Correct and consistent with direct experimental localisation in human, mouse and zebrafish; the human protein legitimately appears among the descendant evidence for the PAINT node. |
| GO:0005743 mitochondrial inner membrane | IDA PMID:32161263 Mitochondrial peptide BRAWNIN is essential for vertebrate re... | ACCEPT | Summary: Carbonate extraction, differential digitonin extraction and protease protection place BRAWNIN in the inner membrane with its C terminus in the intermembrane space. Reason: Core location, directly demonstrated and independently reproduced (PMID:34969438). Supporting Evidence: PMID:32161263 We first established that BR is membrane-bound by sodium carbonate extraction (Fig. 2k). Next, using differential detergent extraction, we determined that BR resides in the IMM and not in the outer mitochondrial membrane (OMM) |
| GO:0005743 mitochondrial inner membrane | IDA PMID:34969438 Defining the interactome of the human mitochondrial ribosome... | ACCEPT | Summary: Independent confirmation of inner-membrane localisation and IMS-facing C terminus, in the context of the early complex III assembly intermediate. Reason: Core location; the assembly intermediate UQCC6 joins (with UQCC1, UQCC2 and SMIM4) is an inner-membrane complex. Supporting Evidence: PMID:34969438 These results confirmed that C12ORF73 represents a protein of the IMM that exposes its C-terminus into the IMS, which agrees with previous studies (Zhang et al., 2020). |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping of the UniProt subcellular location (Mitochondrion inner membrane), itself grounded in two experimental papers. Reason: Correct; duplicates the experimental annotations. |
| GO:0033617 mitochondrial respiratory chain complex IV assembly | IDA PMID:34969438 Defining the interactome of the human mitochondrial ribosome... | MARK AS OVER ANNOTATED | Summary: Based on a slight reduction of cytochrome c oxidase (together with complex I and ATP synthase) after siRNA knockdown of C12ORF73 in HEK293T cells. Reason: The full text of PMID:34969438 was read. The only complex IV observation for C12ORF73 is a slight decrease in steady-state level after knockdown, reported in the same sentence as slight decreases in complex I and complex V; only complex III was significantly reduced, and the paper's own conclusions assign C12ORF73 a role in complex III (cytochrome c reductase) biogenesis only, attributing complex IV assembly to TMEM223. Such a small pleiotropic change is best explained as a secondary consequence of the complex III defect (complex III and IV are co-assembled in supercomplexes). In zebrafish knockouts complex IV activity is not significantly affected (PMID:32161263), and the mouse knockout work summarised by UniProt (PMID:35977508) reports complex IV subunits unaffected. UQCC6 is not shown to do any part of complex IV assembly. Because this is an experimental curation, it is flagged rather than removed. Supporting Evidence: PMID:34969438 Knockdown of C12ORF73 impaired slightly the level of the NADH:ubiquinone oxidoreductase, cytochrome c oxidase, and the ATP synthase; however, the cytochrome c reductase was, similar to SMIM4 knockdown, significantly reduced (Figure 5E). PMID:34969438 We show that TMEM223 is involved in assembly of the cytochrome c oxidase, while SMIM4 and the interacting C12ORF73 contribute to the biogenesis of the cytochrome c reductase. PMID:32161263 while CI, CII, and CIV activities were not significantly affected |
| GO:0034551 mitochondrial respiratory chain complex III assembly | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of complex III assembly function across the BRAWNIN family, grounded in experimental data from human, mouse and zebrafish members. Reason: Core process. The human protein appearing in its own WITH/FROM reflects its experimental annotation being one of the descendant evidences for the PAINT node, which is expected, not circular. |
| GO:0034551 mitochondrial respiratory chain complex III assembly | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the BRAWNIN family signature (IPR027858, Protein BRAWNIN). Reason: The family is defined by this protein and its orthologs, all characterised as complex III assembly factors; the mapping is appropriate. |
| GO:0034551 mitochondrial respiratory chain complex III assembly | IMP PMID:32161263 Mitochondrial peptide BRAWNIN is essential for vertebrate re... | ACCEPT | Summary: Knockdown in human U87MG cells and knockout in zebrafish and mouse embryonic fibroblasts cause loss of assembled complex III dimers and complex III activity. Reason: Core process. Beyond necessity, UQCC6 does part of the work of assembly: it is a component of the early cytochrome b-containing assembly intermediate (with UQCC1, UQCC2 and UQCC5/SMIM4) that binds and stabilises nascent MT-CYB (PMID:34969438, PMID:35977508), so it passes the participation test. A later study found knockdown in human cells to have minimal effect while full deletion impaired complex III assembly (PMID:37769950), consistent with a requirement for only small amounts of the protein. Supporting Evidence: PMID:32161263 we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly PMID:32161263 demonstrating that the requirement for BR in CIII assembly or stability extends to higher vertebrates PMID:34969438 These results demonstrate that C12ORF73FLAG forms a complex with UQCC1, UQCC2, and SMIM4 but not with UQCC3. PMID:37769950 we showed that the deletion rather than knockdown of BRAWNIN impaired the assembly of CIII |
| GO:0034551 mitochondrial respiratory chain complex III assembly | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from mouse Uqcc6 (Q8BTC1), whose knockout causes complete loss of complex III and which is a subunit of the COMB complex that stabilises nascent cytochrome b. Reason: Consistent with direct human data; the mouse study defines the stepwise mechanism (COMA, COMB, COMC) in which UQCC6 acts. Supporting Evidence: PMID:35977508 Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. |
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Download this section (compressed HTML)Q: Should GO add cellular component terms for the mammalian cytochrome b assembly intermediates (COMA, COMB, COMC), so that UQCC6 and UQCC5 can be annotated to COMB membership rather than only to the complex III assembly process?
Suggested experts: Lena Ho, David A Stroud, Peter Rehling
Q: Does UQCC6 have an intrinsic molecular activity (for example a chaperone-like activity toward nascent cytochrome b), or does it act solely as a structural component of the COMB intermediate? Its C terminus faces the intermembrane space, whereas cytochrome b is synthesised at the matrix face.
Q: Why is UQCC6 knockdown in human cells so much milder than knockout, and is this because only small amounts of the protein are needed or because it acts catalytically in successive assembly cycles?
Experiment: Site-specific photo-crosslinking of UQCC6 (transmembrane and IMS-facing residues) in cells with pulse-labelled mitochondrial translation products, followed by identification of crosslinked partners, to test direct contact with MT-CYB versus contact via UQCC1/UQCC2 or UQCC5.
Hypothesis: UQCC6 contacts nascent cytochrome b directly within the membrane.
Type: in organello crosslinking
Experiment: Compare complex IV assembly kinetics (pulse-chase of COX1 into assembled complex IV) in UQCC6-knockout cells with and without complementation of complex III by an alternative route, or against knockouts of other early complex III factors such as UQCC1.
Hypothesis: The complex IV decrease seen after UQCC6 knockdown is secondary to complex III loss.
Type: pulse-chase and BN-PAGE
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