A bifunctional coiled-coil protein generates the membrane-within-condensate architecture of the CO 2 -fixing pyrenoid

Abstract How membranes are integrated into biomolecular condensates is a fundamental question in cell biology. In the algal pyrenoid, an organelle responsible for one-third of global carbon fixation, CO 2 -delivering thylakoid membranes must penetrate a phase-separated condensate of the CO 2 -fixing enzyme Rubisco, but the mechanism governing membrane recruitment into the condensate remains unknown. Here, we demonstrate that the Chlamydomonas reinhardtii protein MITH1 acts as a molecular anchor that brings membrane into the pyrenoid condensate. MITH1 dimerizes into an extended coiled coil with an N-terminal amphipathic helix that binds thylakoid membrane. The coiled coil contains multiple novel binding sites for the Rubisco large subunit, allowing the condensate to wet onto the membrane. The coiled coil extends away from the membrane and promotes membrane organization within the condensate. These findings solve a longstanding mechanistic question in pyrenoid biogenesis and reveal general principles for how membranes are integrated into biological condensates.