Competitive protein recruitment in artificial cells.

Living cells can modulate their response to environmental cues by changing their sensitivities for molecular signals. Artificial cells are promising model platforms to study intercellular communication, but populations with such differentiated behavior remain underexplored. Here, we show the affinity-regulated exchange of proteins in distinct populations of coacervate-based artificial cells via protein-protein interactions (PPI) of the hub protein 14-3-3. By loading different coacervates with different isoforms of 14-3-3, featuring varying PPI affinities, a client peptide is directed to the more strongly recruiting coacervates. By switching affinity of client proteins through phosphorylation, weaker binding partners can be outcompeted for their 14-3-3 binding, inducing their release from artificial cells. Combined, a communication system between coacervates is constructed, which leads to the transport of client proteins from strongly recruiting coacervates to weakly recruiting ones. The results demonstrate that affinity engineering and competitive binding can provide directed protein uptake and exchange between artificial cells.

Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.