Phase-separating pyrenoid proteins form complexes in the dilute phase.

Area of Science:

• Biomolecular condensates
• Phase separation
• Cell biology

Background:

• Most research on biomolecular phase separation overlooks the dilute phase.
• Theoretical models predict stable complexes in the dilute phase, but experimental evidence is lacking.
• Understanding dilute phase behavior is crucial for comprehending condensate formation and function.

Purpose of the Study:

• To experimentally investigate the existence and properties of protein complexes in the dilute phase of phase-separating systems.
• To explore the role of these complexes in the context of the algal pyrenoid, a phase-separated organelle.

Main Methods:

• In vitro reconstitution of the algal pyrenoid using purified Rubisco and EPYC1 proteins.
• Fluorescence Correlation Spectroscopy (FCS) to measure diffusion coefficients and infer complex formation.
• Development of an analytical model to interpret experimental findings.

Main Results:

• Experimental evidence confirms the formation of stable protein complexes in the dilute phase, independent of condensate presence.
• The majority of these dilute-phase complexes consist of a single Rubisco molecule bound to EPYC1.
• The developed analytical model successfully replicates experimental data, offering molecular insights into dilute phase organization.

Conclusions:

• Protein complexes demonstrably exist and are stable within the dilute phase of biomolecular condensates.
• These dilute-phase complexes, particularly those involving Rubisco, likely influence the stability, dynamics, and regulation of condensates.
• The findings necessitate a revised understanding of phase separation, incorporating the significant role of the dilute phase.