A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere''s composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO₂-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO₂. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO₂. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.Rubisco, the most abundant enzyme in the biosphere (1), fixes CO₂ into organic carbon that supports nearly all life on Earth (2, 3). Over the past 3 billion y, the enzyme became a victim of its own success as it drew down the atmospheric CO₂ concentration to trace levels (4) and as the oxygen-producing reactions of photosynthesis filled our atmosphere with O₂ (4). In today’s atmosphere, O₂ competes with CO₂ at Rubisco''s catalytic site, producing the toxic compound phosphoglycolate (5). Phosphoglycolate must be metabolized at the expense of energy and loss of fixed carbon and nitrogen (6). To overcome Rubisco''s limitations, many photosynthetic organisms have evolved carbon-concentrating mechanisms (CCMs) (7, 8). CCMs increase the CO₂ concentration around Rubisco, decreasing O₂ competition and enhancing carbon fixation.At the heart of the CCM of eukaryotic algae is an organelle known as the pyrenoid (9). The pyrenoid is a spherical structure in the chloroplast stroma, discovered more than 130 y ago (10–12). Pyrenoids have been found in nearly all of the major oceanic eukaryotic primary producers and mediate ∼28–44% of global carbon fixation (SI Appendix, Table S1) (3, 13–17). A pyrenoid typically consists of a matrix surrounded by a starch sheath and traversed by membrane tubules continuous with the photosynthetic thylakoid membranes (18). This matrix is thought to consist primarily of tightly packed Rubisco and its chaperone, Rubisco activase (19). In higher plants and non–pyrenoid-containing photosynthetic eukaryotes, Rubisco is instead soluble throughout the chloroplast stroma. The molecular mechanism by which Rubisco aggregates to form the pyrenoid matrix remains enigmatic.Two mechanisms for Rubisco accumulation in the pyrenoid have been proposed: (i) Rubisco holoenzymes could bind each other directly through hydrophobic residues (20), or (ii) a linker protein may link Rubisco holoenzymes together (18, 20). The second model is based on analogy to the well-characterized prokaryotic carbon concentrating organelle, the β-carboxysome, where Rubisco aggregation is mediated by a linker protein consisting of repeats of a domain resembling the Rubisco small subunit (21). Here we find that Rubisco accumulation in the pyrenoid of the model alga Chlamydomonas reinhardtii is mediated by a disordered repeat protein, which we term Essential Pyrenoid Component 1 (EPYC1). Our findings suggest a mechanism for aggregation of Rubisco in the pyrenoid matrix, and highlight similarities and differences between the mechanisms of assembly of the eukaryotic and prokaryotic organelles.