Ubiquitin-based protein interaction assays and related compositions

A kit for detecting a protein substrate for an E3-mediated ubiquitination, said kit comprising:

• i. a first expression construct including a coding sequence for a first output-inducing peptide and a ligation site flanking an end of said first output-inducing peptide coding sequence for ligating a coding sequence of a bait polypeptide sequence in frame with said first output-inducing peptide coding sequence to produce a bait fusion protein, said first expression construct operably linked to a first transcriptional regulatory element;
• ii. a second expression construct including a coding sequence for a second output-inducing peptide and a ligation site flanking an end of said second output-inducing peptide coding sequence for ligating a coding sequence of a prey polypeptide sequence in frame with said output-inducing peptide coding sequence to produce a prey fusion protein, said second expression construct operably linked to a second transcriptional regulatory element;
• and
• iii. a nucleic acid comprising a coding sequence for said E3 operably linked to a third transcriptional regulatory element.

A host cell comprising:

• i. a first nucleic acid encoding said E3 protein,
• ii. a second nucleic acid encoding a bait fusion protein comprising a bait polypeptide sequence fused to a first output-inducing polypeptide;
• and
• iii. a third nucleic acid encoding a prey fusion protein comprising a prey polypeptide sequence fused to a second output-inducing polypeptide,
• wherein said E3 mediates covalent attachment of the bait polypeptide to a prey polypeptide that is a protein substrate of said E3, and wherein physical proximity of said first and second output-inducing polypeptides induces an output signal.