id: A6Q0K5 gene_symbol: CP12 product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:3055 label: Chlamydomonas reinhardtii description: 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. alternative_records: - id: A0A2K3DI03 relationship: REFERENCE_PROTEOME_EQUIVALENT reference_proteome: UP000006906 reviewed_on: '2026-10-19' reason: Retain A6Q0K5 as the primary Swiss-Prot accession. All 107 residues of the Swiss-Prot protein align identically to the 131-residue reference protein, which has a 24-residue extension. Both records share RefSeq XP_001694345.2, supporting the counterpart assignment independently of the sequence match. The precursor boundary difference is explicit; the sequences are not literally identical. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 18,840 sequences across UP000006906 at E <= 1e-5 and sensitivity 7.5, returning 1 hits. Every returned sequence was globally aligned using BLOSUM62 with gap-open -10 and extension -0.5. Scores and sequences for all 1 returned candidates are retained, with 1 detailed alignment files. The heuristic search does not establish genomic absence or unique locus identity. supported_by: - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'target_sequence_count: 18840' - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'candidate_count: 1' - type: SEQUENCE_COMPARISON description: 'A0A2K3DI03: 107 identities across 131 global-alignment columns (81.6794% identity including gaps), 0 substitutions and 24 gap columns. Query coverage is 100.0%; target coverage is 81.6794%. Sequence similarity is interpreted in the manual rationale. Identity over paired residues is 100.0000% (107/107); gap columns are excluded from this denominator.' supported_by: - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: |- query_length: 107 target_length: 131 alignment_columns: 131 identities: 107 mismatches: 0 gap_columns: 24 identity_percent: 81.6794 query_coverage_percent: 100.0 target_coverage_percent: 81.6794 - type: PROTEOME_MEMBERSHIP description: The primary accession is outside every searched exact-taxon reference proteome. Current UniProt records place A0A2K3DI03 in UP000006906 and give it the same taxon as the primary. supported_by: - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'alternative_in_reference_proteome: true' - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'same_taxon: true' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 11-NOV-2015, entry version 31.: UP000006906; 07-OCT-2020, entry version 49.: no Proteomes cross-reference; 27-NOV-2024, entry version 65.: no Proteomes cross-reference. These dated observations do not establish uninterrupted membership/absence, the exact change date, or UniProt''s motivation.' supported_by: - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: |- entry_date: 11-NOV-2015, entry version 31. proteomes: - UP000006906 - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: |- entry_date: 07-OCT-2020, entry version 49. proteomes: [] - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: |- entry_date: 27-NOV-2024, entry version 65. proteomes: [] - type: LOCUS_MAPPING description: Both records carry RefSeq XP_001694345.2, supporting the assignment despite different N-terminal lengths. supported_by: - reference_id: file:CHLRE/CP12/CP12-record-evidence.yaml supporting_text: 'id: XP_001694345.2' existing_annotations: - term: id: GO:0009507 label: chloroplast evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: 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. action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: CP12 is an 8.5-kDa nuclear-encoded chloroplast protein - reference_id: PMID:16259044 supporting_text: The small chloroplast protein CP12 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:24863370 qualifier: enables review: 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. action: MODIFY 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_replacement_terms: - id: GO:0019899 label: enzyme binding supported_by: - reference_id: PMID:24863370 supporting_text: we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments - reference_id: PMID:12846565 supporting_text: oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:24863370 qualifier: enables review: 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. action: MODIFY 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_replacement_terms: - id: GO:0019899 label: enzyme binding supported_by: - reference_id: PMID:24863370 supporting_text: we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:12846565 qualifier: enables review: 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). action: MODIFY 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_replacement_terms: - id: GO:0019899 label: enzyme binding supported_by: - reference_id: PMID:12846565 supporting_text: 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). - term: id: GO:0009507 label: chloroplast evidence_type: IDA original_reference_id: PMID:12846565 qualifier: located_in review: 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. action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: CP12 is an 8.5-kDa nuclear-encoded chloroplast protein - term: id: GO:0019899 label: enzyme binding evidence_type: IPI original_reference_id: PMID:12846565 qualifier: enables review: 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).' action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: 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 - reference_id: PMID:24863370 supporting_text: We quantify a hydrodynamic radius of 3.4 ± 0.2 nm for the CP12 protein with an increase up to 5.2 ± 0.3 nm upon complex formation with GAPDH and PRK - term: id: GO:0009507 label: chloroplast evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: located_in review: 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. action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: CP12 is an 8.5-kDa nuclear-encoded chloroplast protein - term: id: GO:0032991 label: protein-containing complex evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: part_of review: 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. action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12 - term: id: GO:0080153 label: negative regulation of reductive pentose-phosphate cycle evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in review: 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 — 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.' action: ACCEPT 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. supported_by: - reference_id: PMID:12846565 supporting_text: oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex - reference_id: PMID:16259044 supporting_text: 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. - reference_id: PMID:35269851 supporting_text: 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 - reference_id: PMID:35269851 supporting_text: in the ∆CP12-Cr cell extracts, the NADPH-dependent activity of GAPDH displayed a linear curve regardless of DTT - term: id: GO:0005507 label: copper ion binding evidence_type: IDA original_reference_id: PMID:16259044 qualifier: enables review: 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. action: KEEP_AS_NON_CORE 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. supported_by: - reference_id: PMID:16259044 supporting_text: 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 - reference_id: PMID:16259044 supporting_text: 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 - term: id: GO:0016151 label: nickel cation binding evidence_type: IDA original_reference_id: PMID:16259044 qualifier: enables review: 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. action: KEEP_AS_NON_CORE 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. supported_by: - reference_id: PMID:16259044 supporting_text: 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 - reference_id: PMID:16259044 supporting_text: 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 - term: id: GO:0050821 label: protein stabilization evidence_type: IMP original_reference_id: PMID:35269851 review: 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 (ΔCP12 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. action: NEW 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. supported_by: - reference_id: PMID:35269851 supporting_text: Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner - reference_id: PMID:35269851 supporting_text: in the presence of CP12, the addition of DTT restored the initial PRK activity, indicating that CP12 prevented this irreversible inactivation - reference_id: PMID:35269851 supporting_text: a significant difference was observed for PRK amount, with a 6.5-fold decrease of PRK amount in ΔCP12-Cr compared to the WT-Cr strain core_functions: - description: Redox-dependent scaffold/linker that binds GAPDH and PRK to assemble an inactive PRK/GAPDH/CP12 supramolecular complex, negatively regulating the Calvin-Benson-Bassham cycle in the dark molecular_function: id: GO:0019899 label: enzyme binding directly_involved_in: - id: GO:0080153 label: negative regulation of reductive pentose-phosphate cycle locations: - id: GO:0009507 label: chloroplast in_complex: id: GO:0032991 label: protein-containing complex supported_by: - reference_id: PMID:12846565 supporting_text: 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). - reference_id: PMID:24863370 supporting_text: We quantify a hydrodynamic radius of 3.4 +/- 0.2 nm for the CP12 protein with an increase up to 5.2 +/- 0.3 nm upon complex formation with GAPDH and PRK - reference_id: PMID:35269851 supporting_text: the abundance of PRK and its specific activity were significantly reduced in deltaCP12, as revealed by relative quantitative proteomics references: - id: file:CHLRE/CP12/CP12-record-evidence.yaml title: Compact archived record and sequence evidence for A6Q0K5 - id: GO_REF:0000024 title: Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: PMID:12846565 title: 'The small protein CP12: a protein linker for supramolecular complex assembly.' findings: - statement: Oxidized CP12 acts as a redox-sensitive linker for assembly of the PRK/GAPDH/CP12 supramolecular complex; reduced CP12 cannot reconstitute the complex. supporting_text: oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex - statement: The complex has a stoichiometry of 2 PRK dimers, 2 GAPDH tetramers, and CP12. CP12 first binds GAPDH, then PRK is recruited. supporting_text: It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12 - statement: Oxidized CP12 is mainly alpha-helical and flexible; reduced CP12 is mainly unstructured, consistent with intrinsically disordered protein properties. supporting_text: Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured - statement: CP12 is a nuclear-encoded chloroplast protein with a transit peptide confirmed by N-terminal sequencing of the mature protein (residues 28-32). supporting_text: CP12 is an 8.5-kDa nuclear-encoded chloroplast protein - id: PMID:16259044 title: 'Mass spectrometric analysis of the interactions between CP12, a chloroplast protein, and metal ions: a possible regulatory role within a PRK/GAPDH/CP12 complex.' findings: - statement: CP12 specifically binds Cu2+ (Kd = 26 uM) and Ni2+ (Kd = 11 uM) but not Fe2+ or Zn2+, as shown by ESI-MS. supporting_text: 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 - statement: Cu2+ catalyzes re-oxidation of reduced CP12, promoting formation of the disulfide bonds required for linker activity. supporting_text: Cu2+ catalyzes the re-formation of the disulfide bonds of the reduced CP12, leading to recovery of the fully oxidized CP12 - statement: CP12 shows sequence similarity to copper chaperones from Arabidopsis thaliana by hydrophobic cluster analysis, suggesting in vivo relevance of metal binding. supporting_text: 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 - id: PMID:24863370 title: Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy. findings: - statement: FCS confirms CP12 binding to GAPDH and PRK, with hydrodynamic radius increasing from 3.4 nm (free CP12) to 5.2 nm upon complex formation. supporting_text: We quantify a hydrodynamic radius of 3.4 ± 0.2 nm for the CP12 protein with an increase up to 5.2 ± 0.3 nm upon complex formation with GAPDH and PRK - statement: N-terminal and C-terminal cysteine mutants show different structural behavior during unfolding, suggesting distinct roles for the two disulfide bonds in mediating GAPDH vs PRK binding. supporting_text: The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins - id: PMID:35269851 title: Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants. findings: - statement: CRISPR-Cas9 knockout of CP12 in C. reinhardtii results in reduced PRK abundance and activity but normal GAPDH levels and near-normal growth. supporting_text: the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics - statement: CP12 protects PRK from irreversible inactivation in a redox-independent manner, suggesting functions beyond Calvin cycle regulation. supporting_text: in the presence of CP12, the addition of DTT restored the initial PRK activity, indicating that CP12 prevented this irreversible inactivation - id: PMID:24523724 title: 'The CP12 protein family: a thioredoxin-mediated metabolic switch?' findings: - statement: Review establishing CP12 as a conserved redox-sensitive protein found across all oxygenic phototrophs whose only clearly defined function is thioredoxin-mediated regulation of the Calvin cycle via GAPDH/PRK/CP12 complex assembly/disassembly. supporting_text: The only clearly defined function for CP12 in any organism is in the thioredoxin-mediated regulation of the Calvin-Benson cycle - statement: CP12 may have broader regulatory roles beyond Calvin cycle regulation, with evidence suggesting involvement in stress responses and additional metabolic functions. supporting_text: CP12 may have a broader role in the regulation of metabolism, over and above the well established role of CP12 in the regulation of the Calvin-Benson cycle - id: PMID:30923119 title: Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle. findings: - statement: Crystal structures of PRK from both Arabidopsis and Chlamydomonas reveal that the regulatory cysteines are connected by a flexible clamp loop unique to eukaryotic PRKs, completing the structural understanding of all redox-regulated Calvin cycle enzymes. supporting_text: the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements - id: PMID:39036361 title: The primary carbon metabolism in cyanobacteria and its regulation. findings: - statement: Review positioning CP12 as a principal redox regulator of the Calvin cycle especially in cyanobacteria, noting new metabolomics evidence that CP12 variants affect acclimation to external glucose under diurnal conditions and CO2 fluctuations. supporting_text: New results of metabolomic and redox level analyses on strains with Cp12 variants extend the known role of Cp12 regulation towards the acclimation to external glucose supply under diurnal conditions as well as to fluctuations in CO2 levels in the light - id: PMID:37298260 title: Conformational Disorder Analysis of the Conditionally Disordered Protein CP12 from Arabidopsis thaliana in Its Different Redox States. findings: - statement: SAXS analysis of Arabidopsis CP12 confirms it is a conditionally disordered protein; reduced CP12 is highly disordered while oxidized CP12 gains partial structural order through disulfide bond formation but remains flexible. supporting_text: a small angle X-ray scattering (SAXS) analysis of recombinant Arabidopsis CP12 (AtCP12) in a reduced and oxidized form confirmed the highly disordered nature of this regulatory protein - id: PMID:37719215 title: Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii. findings: - statement: PRK is present in moderate excess in C. reinhardtii; approximately 86% of wild-type PRK content is sufficient for full photoautotrophic growth, and overexpression does not increase growth. supporting_text: Immunoblot and growth assays revealed that a PRK content of ≈86% is sufficient to fully restore photoautotrophic growth - id: PMID:31570616 title: Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria. findings: - statement: Cryo-EM structure of the cyanobacterial GAPDH-CP12-PRK ternary complex reveals that CP12's N-terminal disulfide-stabilized helical hairpin plugs the PRK active-site cleft, while the C-terminal region binds GAPDH and influences substrate accessibility of all GAPDH active sites. supporting_text: CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes - statement: CP12 integrates responses from both the redox state (via disulfide bonds) and metabolite availability (NAD(H)/NADP(H) cofactor preference) to coordinate inhibition of GAPDH and PRK in the dark. supporting_text: Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation proposed_new_terms: - proposed_name: PRK-GAPDH-CP12 complex proposed_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). proposed_parent: id: GO:0032991 label: protein-containing complex supported_by: - reference_id: PMID:12846565 supporting_text: It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12 suggested_questions: - question: 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? experts: - Gontero B - Launay H - question: 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)? experts: - Gontero B suggested_experiments: - hypothesis: CP12 copper binding is relevant in vivo for Calvin cycle regulation description: 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 ΔCP12 strains. Use immunoprecipitation to assess complex formation kinetics during light-to-dark transitions. - hypothesis: CP12 has additional interaction partners beyond GAPDH and PRK description: 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.