Composition can buffer protein dynamics within liquid-like condensates.

Abstract

Compartmentalization of biochemistry in membrane-less condensates is a general mode of intracellular organization. In cells, many condensates have complex composition and form via liquid-liquid phase-separation. Rates of biochemical reaction within condensates depend on diffusion rate of components. It remains unclear if condensates can deliver robust functional output despite changing conditions e.g. short-term effects of chemical modifications, intrinsic mutational changes, accumulation of high local concentration of sticky molecules. Here, we address the mechanism that regulates diffusion rate of a scaffold protein (PGL-3) within the liquid-like P granule condensate in adult gonads of C. elegans. Scaffold proteins contribute to assembly of most liquid-like condensates and account for a large fraction of its mass. Using mutational analysis and biophysical perturbations, we generated partially-denatured PGL-3 constructs that phase-separate into condensates in vitro with significantly slower diffusion rates compared to wild-type PGL-3. In contrast, within the P granule condensate (a complex mixture of RNA and >70 proteins) in vivo, these PGL-3 constructs diffuse at rates similar to wild-type PGL-3. This suggests that, in liquid-like condensates, composition can buffer diffusion rates of scaffold proteins (hereafter called ‘dynamics-buffering’). We investigated the underlying mechanism using in vitro reconstitution. It is thought that chaperone proteins and/or ATP-dependent RNA helicases counteract the slow-down of diffusion rate in biomolecular condensates resulting from denaturation of components or inter-polypeptide/RNA chain entanglements. In contrast, we found that weak interactions among two or more components within P granules can account for dynamics-buffering. As expected of a buffer, we found that diffusion rate of PGL-3 constructs remains relatively unchanged regardless of changes in intra-condensate concentration. Once buffering capacity is exhausted i.e. above a threshold intra-condensate concentration, diffusion rate of PGL-3 constructs slow down. We speculate that dynamics-buffering may contribute to robust functional output of cellular liquid-like compartments.