The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

The eukaryotic algal CO2-concentrating mechanism (CCM) is based on a Rubisco-rich organelle called the pyrenoid, which is typically traversed by a network of thylakoid membranes. BST4 is a bestrophin-like transmembrane protein that has previously been identified in the model alga Chlamydomonas reinhardtii as a putative tether that could link the traversing thylakoid membrane network to the Rubisco matrix. In the present study, we show that BST4 forms a higher order complex assembly that localizes to the thylakoid network within the pyrenoid. However, investigation of a bst4 knock-out mutant in Chlamydomonas showed that the absence of BST4 did not result in a CCM-deficient phenotype and that BST4 is not necessary for the formation of the trans-pyrenoid thylakoids. Furthermore, heterologous expression of BST4 was not sufficient to facilitate the incorporation of thylakoids into a reconstituted Rubisco condensate in the land plant Arabidopsis. Subsequent analyses revealed that bst4 was under oxidative stress and showed enhanced non-photochemical quenching associated with CO2 limitation and over acidification of the thylakoid lumen. We conclude that the primary role of BST4 is not as a tethering protein, but rather as an ion channel involved in pH regulation in pyrenoid-based CCMs.