The carbonic anhydrase gene families of Chlamydomonas reinhardtii

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3-. Aquatic photosynthetic organisms have evolved different forms of CO2-concentrating mechanisms to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CO2-concentrating mechanisms is the critical roles played by various specially localized extracellular and intracellular CAs. There are three evolutionarily unrelated CA families designated alpha-, beta-, and gamma-CA. In the green alga, Chlamydomonas reinhardtii Dangeard, eight CAs have now been identified, including three alpha-CAs and five beta-CAs. In addition, C. reinhardtii has another CA-like gene, Glp1 that is similar to known gamma-CAs. To characterize these different CA isoforms, some of the CA genes have been overexpressed to determine whether the proteins have CA activity and to generate antibodies for in vivo immunolocalization. The CA proteins Cah3, Cah6, and Cah8, and the gamma-CA-like protein, Glp1, have been overexpressed. Cah3, Cah6, and Cah8 have CA activity, but Glp1 does not. At least two of these proteins, Cah3 and Cah6, are localized to the chloroplast. Using immunolocalization and sequence analyses, we have determined that Cah6 is located to the chloroplast stroma and confirmed that Cah3 is localized to the chloroplast thylakoid lumen. Activity assays show that Cah3 is 100 times more sensitive to sulfonamides than Cah6. We present a model on how these two chloroplast CAs might participate in the CO2-concentrating mechanism of C. reinhardtii.