PHASE-SEPARATING PYRENOID PROTEINS FORM COMPLEXES IN THE

Abstract

How living cells regulate biomolecular phase separation into a condensed and a dilute phase remains a central question. While most studies have focused on the condensed phase, relatively little is known about the dilute phase. Theoretical studies have suggested that phase separation can be impacted by the formation of stable complexes in the dilute phase of two- component phase-separating systems; however, the existence of such complexes has not been interrogated experimentally. Here we show that such heterocomplexes exist in the dilute phase, using an in vitro reconstitution system of a phase-separated organelle, the pyrenoid, from the model alga Chlamydomonas reinhardtii. The reconstitution system consists of two highly abundant pyrenoid proteins, the CO2-fixing enzyme Rubisco and its linker protein EPYC1. We use fluorescence correlation spectroscopy to measure the dilute-phase diffusion coefficients of these proteins, distinguishing between bound and unbound states. We find that protein complexes consisting of Rubisco and EPYC1 form in the dilute phase with or without condensates present. The majority of these complexes contain one Rubisco molecule, and the fraction of dilute-phase EPYC1 in complex with Rubisco depends on the overall Rubisco:EPYC1 concentration ratio. A minimal model agrees with the measured phase diagram and supports the existence of these Rubisco:EPYC1 complexes. Our results demonstrate the existence of protein oligomers in the dilute phase, which could play a significant role in the dynamics and regulation of condensate assembly.