Controllable protein phase separation and modular recruitment to form responsive membraneless organelles

Benjamin S Schuster,Ranganath Parthasarathy,Holly Ramage,Jessica G Bermudez,Ellen H Reed,Reese M Caldwell,Craig N Jahnke,Matthew C Good,Daniel A Hammer

doi:10.1038/s41467-018-05403-1

Benjamin S Schuster, Ranganath Parthasarathy + Show 7 more

Open Access

https://doi.org/10.1038/s41467-018-05403-1

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Journal: Nature Communications	Publication Date: Jul 30, 2018
Citations: 285	License type: open-access

Affiliation: University of Pennsylvania

Abstract

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

Highlights

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles
LAF-1 is a member of the DDX3 family of RNA helicases and is found in P granules, which are membraneless organelles involved in specification of the germline in Caenorhabditis elegans (Fig. 1a)
By multimerizing the RGG domain from LAF-1 (RGG-RGG and RGGRGG-RGG; illustrated in Fig. 1b), we find that proteins comprised of multiple RGG domains in series more readily selfassemble into liquid droplets than do ones containing a single RGG

Summary

Introduction

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. We manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells. We characterize a minimal, modular platform for engineering membraneless IDP-based compartments, enzymatically trigger liquid mixing and demixing, and demonstrate programmable cargo recruitment and release. The LAF-1 RGG domain is representative of a class of IDPs that have an upper-critical solution temperature (UCST), remaining soluble at high temperatures and condensing out of solution upon cooling below a critical temperature[11,23]

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