Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.

Approximately one-third of the Earth's photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid's three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.