Controllable Protein Phase Separation and Modular Recruitment to Investigate Biochemical Compartmentalization in Membraneless Organelles

Abstract

Many intrinsically disordered proteins (IDPs) self-assemble via liquid-liquid phase separation into spherical droplets, which function as membraneless organelles with critical cellular functions. Despite significant interest, fundamental questions remain unanswered about the functional capabilities of membraneless organelles. It is well established that membrane-bound organelles provide aqueous compartments adapted for specific biochemical reactions, but it is unclear what roles membraneless organelles play in regulating the kinetics of intracellular reactions. The field has been hindered because few tools exist to systematically investigate the consequences of localizing enzymes and substrates to membraneless organelles. Therefore, we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to demonstrate controllable phase separation and cargo recruitment to a synthetic membraneless compartment. First, we demonstrated methods to control phase behavior by externally triggering droplet assembly and disassembly. This was accomplished by using specific proteases to manipulate the valency of IDP domains and presence of solubility-enhancing domains. Second, we characterized permeability of these compartments to soluble macromolecules and devised strategies to target and colocalize cargo molecules into the droplets. Soluble cargos were recruited using either RGG domains or coiled-coiled interaction domains as recruitment modules, and cargo release was triggered by proteolytic removal of the recruitment domains. Droplet assembly and cargo recruitment were robust and occurred in cytoplasm. Our results using this platform suggest it is now possible to controllably recruit multiple enzymes and substrates to stimulus-responsive membraneless organelles. This system provides a much-needed experimental framework to answer unsolved problems in the biophysics of membraneless organelles and to harness IDP compartments for bioengineering applications.