CAH3 (locus Cre09.g415700) is a nuclear-encoded alpha-type carbonic anhydrase of the green alga Chlamydomonas reinhardtii and the only carbonic anhydrase known in its thylakoid lumen. The 310-residue precursor carries a bipartite transit peptide (stromal targeting followed by a twin-arginine lumen-targeting domain); the mature ~29.5 kDa enzyme is a peripheral, lumenal protein of the thylakoid membranes, protected from protease in intact thylakoids. Crystal structures show a canonical alpha-CA fold with a catalytic zinc held by three histidines, a disulfide bond, and an unusual dimer formed by swapped N-terminal arms. The enzyme interconverts bicarbonate and CO2 and is most active at the mildly acidic pH of the illuminated lumen, so in vivo it is thought to work mainly in the dehydration direction. Its main physiological role is in the CO2-concentrating mechanism: bicarbonate pumped into the chloroplast and across the thylakoid membrane is converted to CO2 in the lumen of the thylakoid tubules that traverse the pyrenoid, and the CO2 diffuses into the surrounding Rubisco matrix. Mutants lacking active CAH3 (cia3, ca-1) over-accumulate inorganic carbon yet cannot supply Rubisco with enough CO2 and fail to grow at air levels of CO2; cah3 mutations also suppress the air-dier phenotype of lcib mutants. Immunolocalization places much of CAH3 in the pyrenoid-traversing thylakoids, an enrichment reported to increase when cells are moved to limiting CO2, when CAH3 is also phosphorylated and its activity rises five- to six-fold; a recent expansion-microscopy study instead finds CAH3 concentrated in the central reticulated tubules at both high and low CO2. A second proposed role is at the donor side of photosystem II, where CAH3 co-purifies with PSII-enriched membranes and is suggested to consume protons released by water oxidation by dehydrating bicarbonate; PSII preparations from cia3 have impaired oxygen evolution, but in intact cells at low inorganic carbon photosynthesis in cia3 is limited by CO2 supply to Rubisco rather than by PSII.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004089 carbonate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: CAH3 is a catalytically active alpha-type carbonic anhydrase. Activity has been shown for the native enzyme in complemented cells, for recombinant protein by membrane-inlet mass spectrometry, and the crystal structure shows the zinc site of an active alpha-CA. In vivo it is thought to act mainly in the dehydration direction (bicarbonate to CO2) in the acidic thylakoid lumen. Reason: The InterPro/UniRule mapping is correct and is backed by direct experimental evidence of CA activity (PMID:9482718, PMID:25617045, PMID:39795314) and by genetics (PMID:9159949, PMID:19074623). This is the core molecular function of the gene product. Supporting Evidence: PMID:25617045 Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. PMID:9482718 Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. PMID:39795314 The recombinant protein Ξ±-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: CAH3 binds a catalytic Zn2+ through three conserved histidines, as in other alpha-carbonic anhydrases; zinc is seen in the CAH3 crystal structures (PDB 4XIW, 4XIX). Reason: Correct and structurally confirmed. Zinc binding is the cofactor-binding aspect of the carbonic anhydrase activity rather than a separate function, but the annotation is accurate and harmless. Supporting Evidence: PMID:25617045 Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Γ
resolution, respectively. PMID:38247801 The sequence of the mature CAH3 protein showed 30β40% identity with Ξ±-CAs and up to 90% within the conserved domains, including three His residues functioning as ligands to a Zn atom. |
| GO:0009543 chloroplast thylakoid lumen | IDA PMID:9482718 A novel alpha-type carbonic anhydrase associated with the th... | NEW | Summary: CAH3 is targeted by a bipartite transit peptide with a twin-arginine lumen-targeting domain and is protected from thermolysin in intact thylakoids, placing the mature enzyme in the thylakoid lumen, where it acts. Reason: No GOA cellular component annotation exists for Q39588. The lumenal location is directly shown by protease protection (PMID:9482718) and is supported by every later localization study, including those that place CAH3 in the lumen of the pyrenoid-traversing thylakoids (PMID:22709623, PMID:42465278). This is the compartment in which CAH3 acts and matches the module annoton. Supporting Evidence: PMID:9482718 The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. PMID:22709623 Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. |
| GO:0160223 pyrenoid tubule | IDA PMID:23139834 Phosphorylation controls the localization and activation of ... | NEW | Summary: Immunogold EM, isolated pyrenoid fractions and expansion microscopy all place a large share of CAH3 in the thylakoid tubules that traverse the pyrenoid (in their lumen). How much of CAH3 is there, and whether this changes with CO2, differs between studies. Reason: GO:0160223 is defined as a membranous structure that extends from the thylakoid membrane into and through the pyrenoid matrix. Three independent methods support CAH3 in these tubules (PMID:22709623, PMID:23139834, PMID:42465278). This is where CAH3 is thought to deliver CO2 to Rubisco. It is added alongside, not instead of, the thylakoid lumen term, because a substantial fraction of CAH3 has also been reported in stromal thylakoids. Supporting Evidence: PMID:23139834 In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. PMID:22709623 Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. PMID:42465278 Like the prior immunodetection studies, we also observed the vast majority of CAH3 signal in the pyrenoid tubules. |
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Download this section (compressed HTML)Q: Is the PSII donor-side role of CAH3 physiologically significant in intact cells, or a property of isolated PSII membranes? Most PSII evidence uses the cia3 allele (transit-peptide lesion) and in vitro preparations, while in vivo gas exchange at low Ci shows no PSII limitation.
Q: Does CAH3 really redistribute into the pyrenoid tubules at low CO2 (immunogold, 19% to 37% of total), or is it constitutively enriched in the reticulated tubules (expansion microscopy)? Is any CAH3 left on PSII-containing stromal thylakoids?
Suggested experts: Jonikas MC, Moroney JV
Q: GO has no process term for a CO2-concentrating mechanism and the water-oxidation process term is obsolete. How should the role of a carbonic anhydrase that delivers CO2 to Rubisco (or protons away from PSII) be captured in GO beyond its molecular function and location?
Q: Which residues are phosphorylated, which kinase acts in the lumen, and does phosphorylation change catalytic rate, dimerization or membrane association?
Experiment: Express CAH3 variants targeted or retained in stromal thylakoids versus tubules (e.g. fused to tubule-specific membrane proteins) in a cah3 null background (e.g. a CLiP insertion or the ca-1 nonsense allele), and measure growth, Ci affinity and PSII oxygen evolution in vivo.
Hypothesis: CAH3 activity in pyrenoid tubules, not near PSII, is what is needed for growth at low CO2.
Type: genetic complementation and physiology
Experiment: Map phosphosites by mass spectrometry of low-CO2 thylakoids, then test phospho-null and phosphomimetic CAH3 for activity and localization by expansion microscopy at high and low CO2.
Hypothesis: Low-CO2 phosphorylation of CAH3 controls its activity and/or tubule enrichment.
Type: proteomics, mutagenesis and imaging
Experiment: Compare PSII oxygen evolution and Mn content in cia3, the ca-1 nonsense allele and a catalytically dead lumen-targeted CAH3 (zinc-ligand histidine mutant).
Hypothesis: The PSII phenotypes of cia3 are partly caused by mistargeted CAH3 precursor rather than loss of lumenal CA activity.
Type: genetics and biophysics
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