Rational Tuning of the Concentration-independent Enrichment of Prion-like Domains in Stress Granules

[Display omitted] •Determinants of PrLD recruitment to SGs have not been fully characterized.•Non-aromatic hydrophobic residues tune PrLD recruitment to SGs.•PrLD concentration has little or no effect on SG recruitment.•PrLDs offer a synthetic biology toolkit for tunable targeting to SGs.•PrLD recruitment to SGs is consistent with concentration-independent partitioning. Stress granules (SGs) are large ribonucleoprotein assemblies that form in response to acute stress in eukaryotes. SG formation is thought to be initiated by liquid–liquid phase separation (LLPS) of key proteins and RNA. These molecules serve as a scaffold for recruitment of client molecules. LLPS of scaffold proteins in vitro is highly concentration-dependent, yet biomolecular condensates in vivo contain hundreds of unique proteins, most of which are thought to be clients rather than scaffolds. Many proteins that localize to SGs contain low-complexity, prion-like domains (PrLDs) that have been implicated in LLPS and SG recruitment. The degree of enrichment of proteins in biomolecular condensates such as SGs can vary widely, but the underlying basis for these differences is not fully understood. Here, we develop a toolkit of model PrLDs to examine the factors that govern efficiency of PrLD recruitment to stress granules. Recruitment was highly sensitive to amino acid composition: enrichment in SGs could be tuned through subtle changes in hydrophobicity. By contrast, SG recruitment was largely insensitive to PrLD concentration at both a population level and single-cell level. These observations point to a model wherein PrLDs are enriched in SGs through either simple solvation effects or interactions that are effectively non-saturable even at high expression levels.