Wu, H.;Garde, A.;Stappen, P.;He, S.;Hennacy, J.;Yang, S.;Yu, Z.;Engel, B.;Jonikas, M.

Approximately one-third of global CO₂ fixation occurs in the algal pyrenoid, a condensate of the CO2-fixing enzyme Rubisco traversed by membranes that supply it with CO₂. In the model alga Chlamydomonas reinhardtii, CO2 is delivered by a specialized central reticulated region of these membranes. Here we define how this region is built and identify RBMP2 as the first factor required for its biogenesis. Loss of RBMP2 prevents reticulated-region formation and impairs pyrenoid-dependent growth. RBMP2 domains perform separable roles in extending membrane tubules toward the pyrenoid center and remodeling them into narrow tubules of the reticulated region. The CO2-releasing carbonic anhydrase CAH3 localizes not only to the reticulated region but also to helical interfaces between tubules and minitubules, which expand in some RBMP2 truncation mutants. Our findings advance the basic understanding of pyrenoid function and establish the pyrenoid as a powerful system for studying membrane remodeling within a phase- separated condensate.