LCIB functions as a carbonic anhydrase: evidence from yeast and Arabidopsis carbonic anhydrase knockout mutants

Chlamydomonas reinhardtii evolved a CO₂-concentrating mechanism (CCM) because of the limited CO₂ in its natural environment. One critical component of the C. reinhardtii CCM is the limiting CO₂ inducible B (LCIB) protein. LCIB is required for acclimation to air levels of CO₂. C. reinhardtii cells with a mutated LCIB protein have an 'air-dier' phenotype when grown in low CO₂ conditions, meaning they die in air levels of CO₂ but can grow in high and very low CO₂ conditions. The LCIB protein functions together with its close homolog in C. reinhardtii, limiting CO₂ inducible C protein (LCIC), in a hexameric LCIB-LCIC complex. LCIB has been proposed to act as a vectoral carbonic anhydrase (CA) that helps to recapture CO₂ that would otherwise leak out of the chloroplast. Although both LCIB and LCIC are structurally similar to βCAs, their CA activity has not been demonstrated to date. We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (∆NCE103) and an Arabidopsis thaliana βCA5 knockout mutant (βca5). We show that different truncated versions of the LCIB protein complement ∆NCE103, while the full length LCIB protein complements βca5 plants, so that both the yeast and plant mutants can grow in low CO₂ conditions.