A nuclear CobW/WW-domain factor represses the CO2-concentrating mechanism in the green alga Chlamydomonas reinhardtii

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthesis when dissolved CO2 is limited, but how this energy-intensive system is suppressed when CO2 levels rise has remained unclear. The CCM consumes 15-30% of photosynthetically-generated ATP, making its regulation critical for cellular energy balance. Here, we identify a nuclear repressor of the CCM in the green alga Chlamydomonas reinhardtii. A pull-down screen for interacting partners of the master activator CCM1/CIA5 revealed an uncharacterized protein that co-purifies with CCM1 even after high-salt washes. This protein, designated CCM1-binding protein 1 (CBP1), combines a CobW/CobW_C GTP-binding metallochaperone module with a WW domain characteristic of protein-protein interactions. CBP1 colocalizes with CCM1 in the nucleus regardless of CO2 conditions, and the two proteins interact in vivo. CRISPR/Cas9-based disruption of CBP1 does not affect growth or CCM induction under CO2 limitation but derepresses 27 of 41 CCM1-dependent low-CO2 inducible genes under high-CO2 conditions. These include the periplasmic and intracellular carbonic anhydrases (CAH1, LCIB) and inorganic carbon transporters/channels (LCIA, LCI1, BST1, BST3). Consistently, cbp1 mutants accumulate higher levels of CAH1 and LCIB proteins and exhibit a 40% increase in inorganic carbon affinity under high-CO2 conditions; this phenotype is rescued by CBP1 complementation or by acetazolamide treatment. These results demonstrate that CBP1 prevents unnecessary CCM activity when CO2 is abundant, acting upstream of both transporter/channel and carbonic anhydrase modules. Our findings suggest a regulatory mechanism potentially linking zinc-dependent protein chemistry to CCM gene repression, providing insights into energy-efficient CO2 sensing in aquatic photosynthetic organisms. Significance statementAlgae flourish by activating a CO2-concentrating mechanism (CCM) when dissolved CO2 is limited but must deactivate it when CO2 levels increase to conserve energy. We have identified the nuclear protein that functions as this long sought "off switch" in the model green alga. Deletion of this protein causes cells to overproduce CCM transporters and enzymes, maintaining CCM activity even under high CO2 conditions, demonstrating its essential role in suppressing the system when carbon is abundant. This finding illuminates how algae balance energy consumption with carbon capture and offers a new target for engineering strains that fix CO2 more efficiently for biofuel production or climate-mitigation technologies.