Yeast-liquid-hybrid (ylh) high-throughput method to uncover protein condensate constituents

A method for identifying protein-protein interactions, comprising:

• forming a first modified yeast strain by:
• introducing a Bait plasmid into a first yeast stain, the Bait plasmid having a nucleic acid sequence encoding a protein of interest (Bait protein) fused to a Gal4 DNA Binding Domain (Gal4-DBD) under control of a first promoter, the first yeast strain having either a MATa or MATalpha allele;
• introducing a nucleic acid sequence encoding a Trans element consisting of a free Bait protein, the free Bait protein being free from fusion to Gal4-DBD, into the first yeast strain either through an episomal plasmid or by genomic integration, which makes a condensate around the Bait-Gal4-DBD protein bound to a target promoter containing a DNA sequence to which the Gal4-DBD binds upstream of a selective marker;
• and
• introducing a library of Prey plasmids, each Prey plasmid including a nucleic acid sequence encoding a Prey protein fused to a Gal4 Activation Domain (Gal4-AD) under control of a promoter, the Prey protein optionally being free of a dimerization domain configured for direct protein binding to the Bait-Gal4-DBD protein;
• plating each the first modified yeast strain using a selective media in which the reporter genes are essential and incubating under incubation conditions configured to facilitate protein phase condensation;
• and
• allowing a putative Prey protein to partition into a droplet or condensate formed by the Bait protein and Trans element, inducing expression of the essential gene, resulting in a viable colony.

A method for drug screening against protein condensates, comprising:

• forming a first modified yeast strain by (i) introducing a Bait plasmid into a first yeast stain and then (ii) introducing a Trans element into the first modified yeast strain by episomal plasmid or genomic integration to enable condensate formation at a first promoter to which a Bait-Gal4-DBD protein binds, the Bait plasmid having a nucleic acid sequence encoding a protein of interest fused to a Gal4 DNA Binding Domain (Gal4-DBD) (a Bait-Gal4-DBD protein), the first strain having either a MATa or MATalpha allele;
• forming a second modified yeast strain by introducing a Prey plasmid into a second yeast strain, the Prey plasmid including the nucleic acid sequence encoding a Prey protein fused to a Gal4 Activation Domain (Gal4-AD) under control of a second promoter that partitions into a condensate formed between the Free Bait and the Bait-Gal4-DBD proteins, the second yeast strain having the other of the MATa or MATalpha allele, where a reporter gene has been genomically integrated into the second modified yeast strain;
• forming a mated yeast by mating the first modified yeast strain and the second modified yeast strain;
• growing the mated yeast into a plurality of multi-well plates or a plurality of trays, each well or tray having a selective media in which the reporter genes are essential, exposing at least one well or tray to a chemical or biological agent, and incubating under incubation conditions configured to facilitate protein phase condensation;
• identifying at least one first well or tray in which cells do not grow or form colonies at the same rate or level as a strain not exposed to the chemical or biological agent, indicating that condensate formation did not occur after incubation or is atypical;
• identifying the chemical or biological agent associated with the at least one first well or tray;
• identifying at least one second well or tray in which cells grow or form colonies at a higher rate or level as the strain not exposed to the chemical or biological agent, indicating that condensate formation is enhanced by the chemical or biological agent after incubation;
• and
• identifying the chemical or biological agent associated with the at least one second well or tray.