Characterising the missing thylakoid bicarbonate transport component in Chlamydomonas reinhardtii CO2 concentrating mechanism

Chlamydomonas reinhardtii often faces the challenge of varying CO2 conditions in its environment. As a result, Chlamydomonas evolved a biophysical CO2 concentrating mechanism (CCM) to enhance the photosynthetic performance by concentrating CO2 around Rubisco. Chlamydomonas Rubisco is packaged in a unique organelle called the pyrenoid that is traversed by a network of thylakoid tubules. The current CCM model suggests that a series of membrane transporters deliver HCO3 - across the plasma membrane, chloroplast envelope and thylakoid membranes into the thylakoid lumen. A carbonic anhydrase at the thylakoid tubule network then converts the accumulated HCO3- to CO2, releasing the concentrated CO2 at the pyrenoid. The HCO3- transport model therefore highlights the requirement of a HCO3- transporter(s) at the thylakoid membrane to allow the chloroplast stromal HCO3 - to reach the thylakoid lumen. However, the identity of the HCO3- transporter at the thylakoid membrane has remained elusive. In this study, a knock-out mutant in BST4 displayed poor growth in low CO2 condition and exhibited reduced Ci affinity. Homology modelling showed that BST4 modelled structure resembles a Cl-/HCO3- channel. The BST4 protein was localised to where the thylakoids converge at the centre of the pyrenoid. BST4 contains an extended C-terminal region with similar disorder pattern and residue profile to Rubisco-linker protein EPYC1. However pyrenoid size in bst4 cells was similar to wild type cells, indicating that the C-terminal might not have a structural role but is most likely involved in localising BST4 to the thylakoid network at the centre of the pyrenoid. The results highlight the possibility of BST4 as the elusive HCO3- transporter critical to complete the CO2 delivery route to the pyrenoid.