A Yeast-Liquid-Hybrid (YLH) screening platform for identifying and modulating protein interactions within condensates

Background

Intracellular protein condensates, often formed through the process of liquid-liquid phase separation by intrinsically disordered proteins (IDPs), have emerged as critical regulators of diverse cellular processes such as signaling, transcription, and stress response. These dynamic, membrane-less organelles compartmentalize biochemical reactions and are implicated in both normal physiology and disease states, including neurodegeneration and cancer. As the biological significance of these condensates becomes increasingly apparent, there is a growing need to systematically identify the protein constituents that participate in or modulate their formation. Understanding the molecular composition of protein condensates is essential for unraveling their functional mechanisms and for developing therapeutic strategies targeting aberrant phase separation.

Despite advances in proteomics and molecular biology, current methods for probing protein-protein interactions, such as the classical yeast-two-hybrid (Y2H) system, are poorly suited to capture the complex, multivalent, and often transient interactions characteristic of phase-separated condensates. The Y2H system is optimized for detecting stable, direct binary interactions, but fails to identify interactions that depend on the collective, weak associations driving phase separation. As a result, proteins that only interact within the context of a condensate or require a specific biophysical environment for association remain undetected. This limitation has hindered comprehensive mapping of condensate constituents and impeded efforts to screen for small molecules that modulate condensate dynamics, highlighting the urgent need for new high-throughput approaches tailored to the unique properties of phase-separated protein assemblies.

Technology

The Yeast-Liquid-Hybrid (YLH) method is an advanced platform for detecting protein interactions that occur specifically within phase-separated condensates in living yeast cells. Building on the classical yeast-two-hybrid (Y2H) system, YLH introduces a unique “Trans element”—a nucleic acid encoding a free Bait protein that is not fused to a DNA-binding domain. This free Bait protein drives the formation of protein condensates, particularly around a DNA-bound Bait protein fused to the Gal4 DNA-binding domain. When a Prey protein, fused to a transcriptional activation domain, partitions into these condensates, it brings the activation domain into proximity with the DNA-binding domain, reconstituting a functional transcription factor. This event activates a reporter gene, enabling high-throughput identification of proteins that can partition into condensates. The system is optimized through careful control of media composition, temperature, and the use of selective markers, allowing for robust and scalable screening.

What sets the YLH method apart is its ability to systematically detect interactions mediated by phase separation—interactions that are typically weak, transient, and multivalent, and thus invisible to traditional Y2H assays. By leveraging the formation of liquid-like condensates, YLH can identify protein constituents of membrane-less organelles and other intracellular bodies formed by intrinsically disordered proteins (IDPs). The inclusion of the Trans element is a key differentiator, as it mimics the natural environment where phase separation occurs, enabling the detection of biologically relevant interactions. Furthermore, the system is adaptable for drug screening, allowing researchers to identify compounds that modulate condensate formation, and is valuable in synthetic biology for engineering novel protein assemblies. This combination of high-throughput capability, specificity for phase-separation-mediated interactions, and versatility for diverse applications makes YLH a powerful and differentiated tool in the study of cellular organization and disease mechanisms.

Benefits

• Enables high-throughput identification of proteins that partition into phase-separated condensates, overcoming limitations of classical yeast-two-hybrid assays.
• Detects weak, transient, and multivalent protein interactions mediated by phase separation and condensate formation in living yeast cells.
• Facilitates screening of chemical compounds that modulate condensate formation, supporting drug discovery for diseases involving pathological protein condensates.
• Allows optimization and control of assay conditions (e.g., temperature, media composition) to confirm phase separation-dependent interactions.
• Supports synthetic biology applications by enabling screening and design of synthetic peptides or proteins that partition into or modulate condensates.
• Provides a robust and adaptable platform for studying intracellular condensate biology and protein interaction networks within membrane-less organelles.

Applications

• High-throughput protein condensate screening
• Drug discovery for condensate diseases
• Synthetic organelle engineering
• Screening synthetic peptides for partitioning

Additional info

The Yeast-Liquid-Hybrid (YLH) system identifies proteins partitioning into phase-separated condensates. It utilizes a “Trans element” (free Bait protein) to induce condensate formation around a DNA-bound Bait-DBD. A Prey-AD protein entering these condensates reconstitutes a transcription factor, activating a reporter gene. This enables high-throughput detection of interactions mediated by condensate formation, addressing limitations of classical Y2H for intrinsically disordered proteins.