Combined Automated Annotation using Multiple IEA Methods
A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.
-
Cloning of CAH3, an intracellular alpha-type carbonic anhydrase associated exclusively with the thylakoid membrane
"Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane."
-
CAH3 is in the thylakoid lumen (protease protection of intact thylakoids)
"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."
-
Complementation of cia-3 with the cah3 gene restores growth at air CO2 and an active alpha-type CA
"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."
-
The cia-3 mutant carries two substitutions in the transit peptide and accumulates an inactive CAH3 polypeptide
"Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide."
Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.
-
The ca-1 high-CO2-requiring mutant carries a nonsense mutation in CAH3 and is complemented by the CAH3 gene, linking CAH3 to the CCM
"The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii"
The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo.
-
In vivo, cia3 at low Ci is limited by CO2 supply to Rubisco, not by PSII function
"Overall, the results demonstrate that under low Ci conditions, the mutant lacks the ability to supply Rubisco with adequate CO(2) for effective CO(2) fixation and is not limited directly by any aspect of PSII function."
-
Conclusion that the thylakoid CA is primarily a CCM component
"We conclude that the thylakoid CA is primarily required for the proper functioning of the CCM at low Ci by providing an ample supply of CO(2) for Rubisco."
Thylakoid lumen carbonic anhydrase (CAH3) mutation suppresses air-Dier phenotype of LCIB mutant in Chlamydomonas reinhardtii.
-
cah3 mutations suppress the lcib air-dier phenotype and raise internal Ci more than 20-fold, placing LCIB downstream of CAH3
"We conclude that LCIB functions downstream of CAH3 in the CO2-concentrating mechanism and probably plays a role in trapping CO2 released by CAH3 dehydration of accumulated Ci."
Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii.
-
Immunogold EM places most CAH3 in the thylakoid membranes that pass through the pyrenoid
"Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid."
-
Model in which CAH3 generates CO2 in the lumen that diffuses into the pyrenoid
"The results support our hypothesis that CO(2) might be generated from HCO(3)(-) by Cah3 in the thylakoid lumen with the following CO(2) diffusion into the pyrenoid, where the CO(2) fixing Rubisco is located."
Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.
-
After transfer to low CO2, CAH3 activity rises 5-6 fold without a change in protein level
"When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold."
-
CAH3 is phosphorylated at low CO2 and becomes partly concentrated in PSII-free pyrenoid-crossing thylakoids
"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."
-
Quantification shows the pyrenoid fraction rising from 19% to 37% of total CAH3; most remains in stromal thylakoids
"In the latter, only 19% of the total Cah3 was associated with the pyrenoid region, while in low-CO2-grown cells 37% of the Cah3 protein was associated with the pyrenoid (Table 1)."
-
Isolated pyrenoids lack D1 but are enriched in CAH3 at low CO2
"These results indicate that Cah3, which is associated with PSII in high-CO2-grown cells [6], [7], is being concentrated in the intrapyrenoid thylakoids, which do not contain PSII, during the acclimation to low CO2 conditions."
Super-resolution expansion microscopy reveals nanoscale protein domains and CO(2)-dependent remodeling of Chlamydomonas pyrenoid-traversing membranes.
-
CAH3 is concentrated on the inner face of the central reticulated region of the pyrenoid-traversing membranes
"These results indicate that CAH3 localizes primarily to the reticulated region of the pyrenoid-traversing membranes."
-
CAH3 localization does not change between high and low CO2
"In our experiments, we observed no change in the localization of CAH3 under high CO2 compared to low CO2 (Figure 6a)."
-
CAH3 appears membrane-associated within the tubule lumen
"When we quantified the distribution of CAH3 across the axis of individual tubules, we observed that CAH3 formed two distinct peaks at the edges of the tubules, indicating that CAH3 is not soluble"
A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution.
-
cia3 thylakoids and PSII particles show impaired water splitting that bicarbonate restores; CAH3 proposed to stabilise the Mn cluster
"We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water-oxidizing complex in a functionally active state."
The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal.
-
In cia3 PSII preparations, O2 evolution is slower and is raised by bicarbonate and further by added CAH3
"The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3."
-
Proposed mechanism, local bicarbonate supply as proton carrier at the Mn complex
"We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier."
Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii.
-
Recombinant CAH3 is most active at slightly acidic pH, as in the illuminated lumen
"Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination."
-
Crystal structure shows an N-terminal arm-swapped dimer and a disulfide bond
"CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases."
Carbonic anhydrase CAH3 supports the activity of photosystem II under increased pH.
-
In PSII membranes, CAH3 activity protects PSII activity at raised pH; excess bicarbonate rescues cia3 preparations
"These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO3- dehydration reaction."
Highly Active Carbonic Anhydrase of the Thylakoid Lumen of Chlamydomonas reinhardtii.
-
Recombinant CAH3 has high CO2 hydration activity and is inhibited by sulfonamide CA inhibitors
"The recombinant protein α-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."
CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.
-
Review of the two proposed roles of CAH3 (CCM and PSII)
"Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex."
-
The cia3 substitutions lie next to the twin-arginine motif and block transport into the lumen
"Interestingly, the presence of two point changes near the twin arginine motif in the case of CAH3 is enough to completely disrupt the transport of the precursor protein from the stroma to the thylakoid lumen."
Thylakoid localized bestrophin-like proteins are essential for the CO(2) concentrating mechanism of Chlamydomonas reinhardtii.
-
Current CCM models place CAH3 as the enzyme converting accumulated bicarbonate to CO2 in the lumen of pyrenoid-traversing thylakoids
"A key aspect of current Chlamydomonas CCM models is that accumulated HCO3− is converted to CO2 by CAH3, a carbonic anhydrase located in the thylakoid lumen (11–13)."
Sensitivity of Photosystem II to Photoinhibition in Chlamydomonas reinhardtii Under Conditions of Decreasing CO(2) Depends on a Luminal Carbonic Anhydrase.
-
After low-CO2 acclimation, cia3 shows greater PSII photoinhibition and D1 degradation
"Compared with the two wild-type strains, the cia3 mutant exhibited greater PSII sensitivity to increased light intensity after acclimation to low CO2."