Phase separation underpins many biologically important processes such as RNA metabolism, signaling and CO2-fixation. However, determining the composition of a phase-separated organelle is often challenging due to their sensitivity to environmental conditions which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial

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... e-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in Chlamydomonas chloroplasts, where proximal proteins are labeled by biotin radicals generated from the TurboID-tagged protein. Through the expression of two core pyrenoid components fused with the TurboID tag, we have generated a high confidence pyrenoid proxiome that contains the majority of known pyrenoid proteins plus a number of novel pyrenoid candidates. Fluorescence protein tagging of 8 previously uncharacterized TurboID-identified proteins showed 7 were localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox sensitive iron-sulfur cluster metabolism. This developed pipeline opens the possibility of investigating a broad range of biological processes in Chlamydomonas especially at a temporally resolved sub-organellar resolution.