High-resolution suborganellar localization of Ca2+-binding protein CAS,a novel regulator of CO2-concentrating mechanism

Many aquatic algae induce a CO₂-concentrating mechanism (CCM) associated with active inorganic carbon transport to maintain high photosynthetic affinity using dissolved inorganic carbon even in low-CO₂ (LC) conditions. In the green alga Chlamydomonas reinhardtii, a Ca²⁺-binding protein CAS was identified as a novel factor regulating the expression of CCM-related proteins including bicarbonate transporters. Although previous studies revealed that CAS associates with the thylakoid membrane and changes its localization in response to CO₂ and light availability, its detailed localization in the chloroplast has not been examined in vivo. In this study, high-resolution fluorescence images of CAS fused with a Chlamydomonas-adapted fluorescence protein, Clover, were obtained by using a sensitive hybrid detector and an image deconvolution method. In high-CO₂ (5% v/v) conditions, the fluorescence signals of Clover displayed a mesh-like structure in the chloroplast and part of the signals discontinuously overlapped with chlorophyll autofluorescence. The fluorescence signals gathered inside the pyrenoid as a distinct wheel-like structure at 2 h after transfer to LC-light condition, and then localized to the center of the pyrenoid at 12 h. These results suggest that CAS could move in the chloroplast along the thylakoid membrane in response to lowering CO₂ and gather inside the pyrenoid during the operation of the CCM.