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

Abstract

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3− from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3− to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3− 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 CO2 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 CO2, 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 CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3− accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Assembly of the algal CO 2 -fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

“…RBMP1 is predicted to be a Ca 2+ -activated anion channel of the bestrophin family ( fig. S2, D and F), several members of which are thought to supply HCO 3 − to the lumen of the tubules for conversion to CO 2 (29). Although the previously described members localized primarily to membranes outside the pyrenoid, RBMP1 localizes exclusively to the tubules themselves, which may allow it to directly feed HCO 3 − to the tubules for conversion to CO 2 .…”

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

“…Intriguingly, RBMP1 is predicted to be a Ca 2+ -activated anion channel of the bestrophin family ( Fig. S2D and S3F), of which several members are thought to supply HCO3to the lumen of the tubules for conversion to CO2 (28). Whereas the previously described members localized primarily to membranes outside the pyrenoid, RBMP1 localizes exclusively to the tubules themselves, which may allow it to directly feed HCO3to the tubules for conversion to CO2.…”

Chloride involvement in the synthesis, functioning and repair of the photosynthetic apparatusin vivo

“…While bestrophin channels in Metazoa are typically considered to be Cl − channels, they are also highly permeable to HCO 3 − (Qu & Hartzell, 2008), and a bestrophin‐like protein is the HCO 3 − channel in the thylakoid membrane of Chlamydomonas reinhardtii that is involved in the CO 2 concentrating mechanism (CCM) (Mukherjee et al , 2019). The bestrophin‐like channels in C. reinhardtii are localised in the thylakoid tubules in the pyrenoid, colocalised with the luminal carbonic anhydrase Cah3, generating CO 2 which diffuses through the thylakoid membrane to Rubisco localized in the pyrenoid (Mukherjee et al , 2019). For C 3 terrestrial flowering plants such as A. thaliana , HCO 3 − influx to the thylakoid lumen would, in the presence of soluble luminal carbonic anhydrase (as in A. thaliana : Ignatova et al , 2019a), act as an uncoupler by reacting with HCO 3 with H + to produce membrane‐permeable CO 2 and H 2 O.…”