CP12 is a small (~8.5 kDa), nuclear-encoded, conditionally disordered chloroplast protein that functions as a redox-sensitive linker/scaffold mediating the assembly of a supramolecular PRK/GAPDH/CP12 complex. In the dark, oxidized CP12 (with two intramolecular disulfide bonds) sequentially binds GAPDH then PRK, forming a ternary complex that inactivates both Calvin cycle enzymes. In the light, thioredoxin-mediated reduction of CP12 disulfide bonds causes the complex to dissociate, releasing active enzymes. CP12 also binds Cu2+ and Ni2+ ions in vitro; Cu2+ catalyzes re-oxidation of CP12 thiols, potentially linking metal homeostasis to Calvin cycle regulation.
Definition: A protein complex consisting of phosphoribulokinase (PRK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the linker protein CP12. The complex forms in the dark under oxidizing conditions and sequesters PRK and GAPDH in an inactive state, thereby negatively regulating the Calvin-Benson-Bassham cycle. Stoichiometry: 2 PRK dimers + 2 GAPDH tetramers + CP12.
Justification: No GO complex term currently exists for this well-characterized supramolecular complex, which is conserved across photosynthetic organisms from cyanobacteria to higher plants. The current annotation uses the generic GO:0032991 (protein-containing complex).
Parent term: protein-containing complex
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009507 chloroplast | IEA GO_REF:0000044 | ACCEPT | Summary: Chloroplast localization is well-supported by multiple lines of evidence. CP12 has a 27-residue chloroplast transit peptide confirmed by direct protein sequencing (PMID:12846565), and IDA evidence from CAFA also supports this localization. This IEA annotation from UniProt subcellular location mapping is consistent with all other evidence. Reason: Although this is an IEA annotation, it is fully consistent with the IDA evidence (PMID:12846565) showing chloroplast localization via transit peptide identification and direct protein sequencing. The UniProt record confirms chloroplast transit peptide residues 1-27 with experimental evidence. Supporting Evidence: PMID:12846565 CP12 is an 8.5-kDa nuclear-encoded chloroplast protein PMID:16259044 The small chloroplast protein CP12 |
| GO:0005515 protein binding | IPI PMID:24863370 Conformational modulation and hydrodynamic radii of CP12 pro... | MODIFY | Summary: This annotation records CP12 interaction with PRK (P19824) based on fluorescence correlation spectroscopy (FCS) experiments (PMID:24863370). While the interaction is real and well-documented, GO:0005515 (protein binding) is too generic per curation guidelines. CP12 binds the enzyme PRK as part of its core regulatory function; GO:0019899 (enzyme binding) is more informative and already annotated. Reason: Per curation guidelines, GO:0005515 (protein binding) is uninformative. CP12's interaction with PRK is its core molecular function -- it acts as a scaffold/linker that binds PRK to form the regulatory PRK/GAPDH/CP12 complex. GO:0019899 (enzyme binding) is already annotated for the GAPDH interaction and is equally appropriate for the PRK interaction, as PRK is an enzyme (phosphoribulokinase, EC 2.7.1.19). Proposed replacements: enzyme binding Supporting Evidence: PMID:24863370 we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments PMID:12846565 oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex |
| GO:0005515 protein binding | IPI PMID:24863370 Conformational modulation and hydrodynamic radii of CP12 pro... | MODIFY | Summary: This annotation records CP12 interaction with GAPDH (P50362) based on FCS experiments (PMID:24863370). Same issue as the PRK protein binding annotation -- GO:0005515 is too generic. The GAPDH interaction is already covered by the enzyme binding annotation from PMID:12846565. Reason: GO:0005515 is uninformative per curation guidelines. CP12's interaction with GAPDH is its core function as a regulatory linker. GO:0019899 (enzyme binding) is more appropriate, as GAPDH is an enzyme (glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.13). This interaction is already annotated as enzyme binding from PMID:12846565. Proposed replacements: enzyme binding Supporting Evidence: PMID:24863370 we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments |
| GO:0005515 protein binding | IPI PMID:12846565 The small protein CP12: a protein linker for supramolecular ... | MODIFY | Summary: This annotation records CP12 interaction with both PRK (P19824) and GAPDH (P50362) based on reconstitution assays and SPR binding studies (PMID:12846565). As with the other protein binding annotations, GO:0005515 is too generic. The interactions are real but better captured by GO:0019899 (enzyme binding). Reason: GO:0005515 is uninformative per curation guidelines. CP12 acts as a linker for two Calvin cycle enzymes; enzyme binding (GO:0019899) is the appropriate term. The original paper clearly demonstrates CP12 binding to both PRK and GAPDH through reconstitution assays and surface plasmon resonance. Proposed replacements: enzyme binding Supporting Evidence: PMID:12846565 oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). |
| GO:0009507 chloroplast | IDA PMID:12846565 The small protein CP12: a protein linker for supramolecular ... | ACCEPT | Summary: Direct experimental evidence for chloroplast localization. The PMID:12846565 study determined the N-terminal sequence of the mature protein (residues 28-32), confirming cleavage of the chloroplast transit peptide and thereby demonstrating chloroplast import. UniProt lists this as transit peptide residues 1-27 with evidence from PMID:12846565. Reason: Strong IDA evidence from direct protein sequencing confirming the transit peptide cleavage site. This is the primary experimental evidence for chloroplast localization and directly demonstrates that CP12 is imported into the chloroplast. Supporting Evidence: PMID:12846565 CP12 is an 8.5-kDa nuclear-encoded chloroplast protein |
| GO:0019899 enzyme binding | IPI PMID:12846565 The small protein CP12: a protein linker for supramolecular ... | ACCEPT | Summary: Enzyme binding is CP12's core molecular function. CP12 acts as a regulatory linker that binds two Calvin cycle enzymes: GAPDH (P50362, glyceraldehyde-3-phosphate dehydrogenase) and PRK (P19824, phosphoribulokinase). The WITH column specifies GAPDH (P50362). This was demonstrated through reconstitution assays and SPR binding studies (PMID:12846565), and independently confirmed by FCS (PMID:24863370). Reason: This is the most informative molecular function term for CP12's core activity. CP12 is defined by its ability to bind and regulate GAPDH and PRK through redox-dependent complex assembly. Enzyme binding accurately captures this scaffold/linker function. The evidence is strong, based on multiple in vitro reconstitution approaches. Supporting Evidence: PMID:12846565 oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change PMID:24863370 We quantify a hydrodynamic radius of 3.4 Β± 0.2 nm for the CP12 protein with an increase up to 5.2 Β± 0.3 nm upon complex formation with GAPDH and PRK |
| GO:0009507 chloroplast | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on sequence similarity to Arabidopsis thaliana CP12 (O22914). Consistent with the IDA evidence from PMID:12846565 and the IEA annotation. This is redundant with the IDA evidence but not incorrect. Reason: Fully consistent with the IDA evidence (PMID:12846565). The ISS transfer from A. thaliana CP12 is appropriate given that chloroplast localization is a conserved feature of all CP12 family members, which universally possess chloroplast transit peptides. Supporting Evidence: PMID:12846565 CP12 is an 8.5-kDa nuclear-encoded chloroplast protein |
| GO:0032991 protein-containing complex | ISS GO_REF:0000024 | ACCEPT | Summary: CP12 is part of the PRK/GAPDH/CP12 supramolecular complex. The annotation to the generic GO:0032991 (protein-containing complex) is correct but very broad. The full complex stoichiometry is 2 PRK dimers + 2 GAPDH tetramers + CP12 (PMID:12846565). A more specific GO complex term would be ideal, but no dedicated term for the PRK/GAPDH/CP12 complex currently exists in GO. Reason: While GO:0032991 is generic, there is no more specific GO term available for the PRK/GAPDH/CP12 complex. The annotation is accurate -- CP12 is indeed a component of a well-characterized protein-containing complex. The ISS transfer from A. thaliana CP12 is appropriate since the PRK/GAPDH/CP12 complex is conserved across photosynthetic organisms. Proposing a new GO term for this complex could be considered. Supporting Evidence: PMID:12846565 It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12 |
| GO:0080153 negative regulation of reductive pentose-phosphate cycle | ISS GO_REF:0000024 | ACCEPT | Summary: This is the core biological process annotation for CP12. CP12 negatively regulates the Calvin-Benson-Bassham (reductive pentose-phosphate) cycle by sequestering PRK and GAPDH into an inactive complex in the dark. The ISS transfer from A. thaliana CP12 (O22914) is well-justified, as this regulatory function is conserved across all characterized CP12 proteins. CRISPR-Cas9 knockout of CP12 in C. reinhardtii (PMID:35269851) provides direct IMP-grade evidence in the target organism β delta-CP12 cells show abolished DTT-dependent regulation of GAPDH activity and 3-fold reduced PRK specific activity, confirming that CP12 is required in vivo for dark regulation of the Calvin cycle. An upgraded experimental evidence annotation (e.g. IMP from PMID:35269851) should be considered by the curation source. Reason: GO:0080153 precisely captures CP12's core biological process function. CP12 coordinates the dark inactivation of GAPDH and PRK, two key Calvin cycle enzymes, by assembling them into an inactive supramolecular complex. This is the most specific and accurate BP term for CP12's role. The ISS annotation is consistent with strong direct experimental evidence in C. reinhardtii from both reconstitution experiments (PMID:12846565) and CRISPR knockout studies (PMID:35269851), so an IMP upgrade in the source GOA is warranted. Supporting Evidence: PMID:12846565 oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex PMID:16259044 a PRK/GAPDH/CP12 complex that is involved in CO2 assimilation in photosynthetic organisms. The redox state of CP12 regulates its role as a protein linker. PMID:35269851 The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes PMID:35269851 in the βCP12-Cr cell extracts, the NADPH-dependent activity of GAPDH displayed a linear curve regardless of DTT |
| GO:0005507 copper ion binding | IDA PMID:16259044 Mass spectrometric analysis of the interactions between CP12... | KEEP AS NON CORE | Summary: IDA evidence from ESI-MS experiments demonstrating specific Cu2+ binding by CP12 with Kd = 26 +/- 1 uM (PMID:16259044). Importantly, Cu2+ also catalyzes re-formation of CP12 disulfide bonds, potentially linking copper to Calvin cycle regulation. The authors note sequence similarity between CP12 and copper chaperones from A. thaliana. However, the primary biological function of CP12 is as a regulatory scaffold, not as a dedicated copper-binding protein; the in vivo significance of copper binding remains uncertain. Reason: The copper binding is real (IDA evidence, specific binding with measured Kd), but it represents a secondary/potential moonlighting function rather than CP12's core evolved function. CP12's primary role is as a redox-dependent linker for the PRK/GAPDH complex. The copper-catalyzed thiol oxidation may be physiologically relevant as it could promote CP12's active (oxidized) conformation, but this remains speculative. Retaining as non-core appropriately reflects the evidence. Supporting Evidence: PMID:16259044 The oxidized protein bound specifically Cu2+ and Ni2+ (Kd of 26+/-1 microM and 11+/-1 microM, respectively); other cations such as Fe2+ and Zn2+ did not bind PMID:16259044 Cu2+ catalyzes the re-formation of the disulfide bonds of the reduced CP12, leading to recovery of the fully oxidized CP12 that is then able to bind a Cu2+ ion |
| GO:0016151 nickel cation binding | IDA PMID:16259044 Mass spectrometric analysis of the interactions between CP12... | KEEP AS NON CORE | Summary: IDA evidence from ESI-MS experiments showing specific Ni2+ binding by CP12 with Kd = 11 +/- 1 uM (PMID:16259044). The binding is more specific than Cu2+ (lower Kd) but the biological relevance is even less clear. Nickel is not known to play a significant role in chloroplast metabolism in green algae. The His74 mutation had no impact on metal binding, suggesting the binding site may involve other residues. Reason: The nickel binding is experimentally demonstrated (IDA) with good specificity (Kd = 11 uM, no binding of Fe2+ or Zn2+), but there is no evidence for an in vivo role. Unlike copper, nickel does not catalyze CP12 disulfide bond formation, so a regulatory connection to CP12's primary function is not established. This is a secondary characteristic best kept as non-core. Supporting Evidence: PMID:16259044 The oxidized protein bound specifically Cu2+ and Ni2+ (Kd of 26+/-1 microM and 11+/-1 microM, respectively); other cations such as Fe2+ and Zn2+ did not bind PMID:16259044 the high similarity between CP12 and copper chaperones from Arabidopsis thaliana, as judged by hydrophobic cluster analysis, provides additional evidence for the relevance of metal binding for the in vivo situation |
| GO:0050821 protein stabilization | IMP PMID:35269851 Reduction in Phosphoribulokinase Amount and Re-Routing Metab... | NEW | Summary: Proposed new annotation reflecting a redox-independent moonlighting function of CP12. PMID:35269851 demonstrates that CP12 stabilizes PRK against irreversible inactivation both in vitro (recombinant CP12 prevents irreversible loss of PRK activity in a redox-independent manner; DTT can restore activity only when CP12 is present) and in vivo (ΞCP12 cells show 6.5-fold reduction in PRK protein abundance with no change in mRNA, consistent with loss of post-translational stabilization). Site-directed mutagenesis identifies specific residues (D36, E39, E40, W35, H47) required for this protective function. Reason: PMID:35269851 provides both in vivo (knockout) and in vitro (recombinant protein + mutagenesis) evidence that CP12 specifically protects PRK from irreversible inactivation in a redox-independent manner. This is mechanistically distinct from CP12's redox-dependent regulatory role in the PRK/GAPDH/CP12 complex and warrants a separate annotation. GO:0050821 (protein stabilization) accurately captures this function as the term covers maintenance of protein structure/integrity and prevention of aggregation/degradation. Supporting Evidence: PMID:35269851 Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner PMID:35269851 in the presence of CP12, the addition of DTT restored the initial PRK activity, indicating that CP12 prevented this irreversible inactivation PMID:35269851 a significant difference was observed for PRK amount, with a 6.5-fold decrease of PRK amount in ΞCP12-Cr compared to the WT-Cr strain |
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Download this section (compressed HTML)Q: Is the copper ion binding by CP12 physiologically relevant in vivo, or is it an in vitro artifact? Does copper-catalyzed re-oxidation of CP12 thiols contribute to dark regulation of the Calvin cycle under normal conditions?
Suggested experts: Gontero B, Launay H
Q: Should CP12's role in protecting PRK from irreversible inactivation (redox-independent) be annotated separately from its role in dark/light regulation of the Calvin cycle (redox-dependent)?
Suggested experts: Gontero B
Experiment: Measure Calvin cycle enzyme activity and PRK/GAPDH/CP12 complex formation in C. reinhardtii cells grown under copper-depleted vs. copper-replete conditions, comparing wild-type and ΞCP12 strains. Use immunoprecipitation to assess complex formation kinetics during light-to-dark transitions.
Hypothesis: CP12 copper binding is relevant in vivo for Calvin cycle regulation
Experiment: Perform in vivo crosslinking mass spectrometry (XL-MS) or proximity labeling (BioID/TurboID) with tagged CP12 in C. reinhardtii to identify the full interactome, including reported interactions with aldolase and malate dehydrogenase.
Hypothesis: CP12 has additional interaction partners beyond GAPDH and PRK
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