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

Abstract: 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 over 130 years ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. To improve our understanding of the pyrenoid, we developed a high-throughput fluorescent protein-tagging pipeline to determine the localizations of 146 candidate proteins in the green alga Chlamydomonas reinhardtii. Combining this localization data with protein-protein interaction data has allowed us to generate a spatial interactome of the pyrenoid. Further characterization of a low complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), shows that it 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 finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose a model where 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. Furthermore, EPYC1 and other newly identified pyrenoid components are being introduced into higher plants with a goal to engineer this organelle to enhance crop carbon-fixation efficiency.