Thylakoid localized bestrophin-like proteins are essential for the CO(2) concentrating mechanism of Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii possesses a CO(2) concentrating mechanism (CCM) that helps in successful acclimation to low CO(2) conditions. Current models of the CCM postulate that a series of ion transporters bring HCO(3)(−) from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO(3)(−) to CO(2), raising the CO(2) concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO(3)(−) transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO(2) conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO(2), indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1–3 were unable to grow at low CO(2) concentrations, exhibited a reduced affinity to inorganic carbon (C(i)) compared with the wild-type cells, and showed reduced C(i) uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO(3)(−) accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.