The yeast two-hybrid system is a powerful tool for detecting protein-protein interactions in living cells. It uses bait and prey proteins fused to DNA-binding and activation domains to create a functional transcription factor when they interact, activating reporter genes.

This technique offers advantages like detecting weak interactions and enabling high-throughput screening. However, it has limitations such as false positives and negatives. Mammalian two-hybrid systems provide an alternative for studying interactions in a more physiologically relevant context for some proteins.

Yeast Two-Hybrid System

Principles of yeast two-hybrid system

• Detects protein-protein interactions in living yeast cells
• Components work together to create functional transcription factor:
  • Bait protein fused to DNA-binding domain (DBD) binds specific DNA sequence
  • Prey protein fused to activation domain (AD) recruits RNA polymerase
  • Reporter gene activated when bait and prey interact (LacZ, HIS3)
• Transcription factor reconstitution occurs when DBD and AD brought together by protein interaction
• Yeast serves as eukaryotic host organism easily manipulated genetically
• Plasmid vectors carry bait and prey fusion constructs with selectable markers (URA3, LEU2)

Detection of protein-protein interactions

• Bait-prey interaction brings DBD and AD into close proximity
• Reconstituted transcription factor binds promoter initiates reporter gene transcription
• Reporter gene expression measured through:
  • Colorimetric assays (X-gal for β-galactosidase)
  • Auxotrophic growth selection on media lacking specific nutrients (histidine)
• Strength of protein interaction correlates with reporter gene expression level
• Positive interactions identified by colony color change or growth on selective media

Advantages vs limitations of two-hybrid system

• Advantages:
  • Detects interactions in living cells revealing physiological relevance
  • High-throughput screening enables large-scale interactome mapping
  • Identifies weak and transient interactions often missed by other methods
  • Cost-effective and technically simple compared to biochemical approaches
• Limitations:
  • False positives arise from auto-activating proteins or non-specific interactions
  • False negatives occur with proteins requiring post-translational modifications
  • Yeast cellular environment may not reflect native conditions for mammalian proteins
  • Nuclear localization requirement excludes some cytoplasmic proteins
  • Membrane proteins often difficult to study due to hydrophobic nature

Yeast vs mammalian two-hybrid systems

• Mammalian system uses mammalian cells as host (HEK293, HeLa)
• Components similar: DNA-binding domain, activation domain, reporter gene
• Key differences:
  • Host cell type affects protein folding and modifications
  • Vectors adapted for mammalian expression with different promoters
  • Reporter genes optimized for mammalian cells (Luciferase, GFP)
• Mammalian system advantages:
  • Native environment for mammalian proteins preserves physiological context
  • Proper post-translational modifications maintained
  • Suitable for membrane proteins and extracellular interactions
• Applications tailored to system strengths:
  • Yeast: Large-scale interactome mapping, drug target identification
  • Mammalian: Validation of interactions in physiological context, study of mammalian-specific complexes
• Choice depends on proteins of interest and research question requiring appropriate cellular context