A Spatial Interactome Reveals the Protein Organization of the Algal CO2 Concentrating Mechanism

One-third of global CO2 fixation is mediated by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2 concentrating mechanism (CCM), built around a mysterious organelle called the pyrenoid. Here, we leveraged new tools to determine the localizations of 146 candidate CCM proteins in the model alga Chlamydomonas reinhardtii. Twelve of the tagged proteins localized to the pyrenoid in six distinct sub-pyrenoid localizations: matrix, membrane tubules, plates, a mesh layer, and two punctate layers. Many stromal proteins can access the pyrenoid matrix. The proteins with access to the matrix tend to be small, suggesting that the pyrenoid may show protein size selectivity. Contrary to current models, the carbonic anhydrase CAH6 is in the flagella. Affinity purification mass spectrometry of 38 CCM-related proteins reveals dozens of additional components, including novel Rubisco interacting proteins, photosystem I assembly factor candidates and inorganic carbon flux components. Together, our spatial interactome reveals new protein components, sub-compartments and biophysical characteristics that open doors to a detailed molecular characterization of the algal CCM.