biostudies-literatureUnknown6(1)He GWhile most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a Communications biology19https://www.ebi.ac.uk/biostudies/studies/S-EPMC9825591biostudies-literaturePhase-separating pyrenoid proteins form complexes in the dilute phase.PMC9825591Jonikas MCGrandPre TWingreen NSWang QHe GZhang YWilson HfalsePhase-separating pyrenoid proteins form complexes in the dilute phase.While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a 2023-01-01T00:00:00Z2023 Jan2025-04-21T14:20:55.874Z2025-02-19T03:30:26.505ZS-EPMC98255913661106210.1038/s42003-022-04373-x