Induction of a high-CO2-inducible, periplasmic protein, H43, and its application as a high-CO2-responsive marker for study of the high-CO2-sensing mechanism in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a broad range of environmental CO2 concentrations. We observed that the cells synthesized a specific 43 kDa protein, H43, in the periplasmic space under photoautotrophic high-CO2 conditions. Under low-CO2 conditions, H43 disappeared. However, H43 mRNA expression was observed even under heterotrophic low-CO2 conditions when the cells were grown with 17.4 mM acetate in darkness. When the cells were treated with 4,4'-dithiocyanatostilbene-2,2'-disulfonate (DIDS) and mersalyl to modify cell surface proteins, H43 mRNA expression was strongly affected under both heterotrophic and photoautotrophic conditions. The H43 induction pattern in a mitochondrial respiration-deficient mutant dum-1 that lacks cytochrome c oxidase was the same, but the level was much lower than that in the wild type. Even under illumination, the dissolved CO2 concentration in the culture rapidly increased slightly following the addition of acetate and dramatically increased even further by the inhibition of photosynthesis with DCMU. Radiotracer experiments with [U-C-14]acetate revealed that (CO2)-C-14 release from cells was greater in darkness than in the light due to the great stimulation of internal CO2 evolution, resulting in an increase in external CO2 concentration. Strong light inhibited H43 induction and DCMU promoted the induction under photoheterotrophic low-CO2 conditions. The results demonstrate that H43 is strictly regulated by a concentration of CO2 resulting from respiration and photosynthesis. Our results suggest that Chlamydomonas induces high-CO2-responsive protein H43 by sensing the concentration of ambient CO2 with the contribution of cell surface protein.