Investigating the eukaryotic CO2-fixing phase-separated organelle, the pyrenoid

In most eukaryotic algae, an organelle called the pyrenoid helps concentrate CO₂ to enhance carbon fixation by the enzyme Rubisco. We recently found that in Chlamydomonas reinhardtii, the pyrenoid has liquid-like behavior including rapid condensation and dissolution during cell division. Our data suggests that the matrix is primarily composed of Rubisco and a linker protein, EPYC1. Rubisco and EPYC1 each have multiple binding sites for the other, allowing the two proteins to form a multivalent phase separation system. We showed that Rubisco and EPYC1 are sufficient to phase separate in an in vitro reconstitution experiment. Here, we measure the phase boundary in terms of concentration of both proteins, which is in the uM range. We use microscale thermophoresis (MST) to determine the dissociation constant (Kd) of EPYC1-Rubisco binding. And we use fluorescence correlation spectrometer (FCS) to determine the protein particle sizes. Combined together, our results suggest the existence of small oligomer complexes composed of EPYC1 and Rubisco in the dilute phase. Our work reveals that the stoichiometry of oligomer complexes is a key factor that regulates phase separation, which might be a general principle in multivalent phase separation system in biology.