Effect of a membrane protein condensation phase transition on reaction-diffusion kinetics.

Abstract

Reversible biomolecular phase transitions, such as liquid-liquid phase transition in cytosolic proteins, and phosphorylation-driven condensation in membrane-bound systems have been cited as underlying mechanisms for numerous important cellular processes such as signal transduction. For example, condensation of the T cell signaling protein LAT, and, separately, Epidermal Growth Factor Receptor has been shown to elongate the membrane dwell time of the Ras-GEF SOS, enabling it to overcome a series of slow, membrane dependent activation steps. However, evidence from in-vitro reconstitution experiments suggests that formation of a condensed phase may not solely be beneficial to membrane-associated enzyme kinetics. SOS is both a scaffolding component, and a downstream effector of signaling condensates upstream of the Ras/Erk pathway. In such systems, its substrate Ras needs to have access to SOS for nucleotide exchange to take place. Sequestration of SOS proteins within a networked condensate may inhibit the ability of Ras to encounter SOS by diffusion. To investigate, we reconstitute protein condensation phase transitions consisting of the phosphotyrosine tails of either LAT or EGFR on supported lipid bilayers, along with the crosslinking components Grb2 and the PR domain of SOS to induce condensation. Under these conditions, single molecule TIRF microscopy can be used to localize and track membrane diffusion and partitioning between the condensed domains and surrounding sparse phase.