Generation and Validation of MYTH Baits: iMYTH and tMYTH Variants

Area of Science:

• Molecular Biology
• Biochemistry
• Yeast Genetics

Background:

• Membrane Yeast Two-Hybrid (MYTH) screening is crucial for identifying protein-protein interactions.
• The generation of effective baits is a critical first step in MYTH screening.
• Different protein characteristics necessitate distinct bait generation strategies.

Purpose of the Study:

• To describe and validate methods for generating baits for MYTH screening.
• To differentiate between integrated MYTH (iMYTH) and traditional MYTH (tMYTH) bait generation.
• To provide a protocol for efficient MYTH bait construction.

Main Methods:

• Integrated MYTH (iMYTH): Endogenous C-terminal tagging of native yeast proteins on the chromosome.
• Traditional MYTH (tMYTH): Ectopic expression of N- or C-terminally tagged proteins from a plasmid.
• Validation of generated iMYTH and tMYTH baits.

Main Results:

• Successful generation and validation of both iMYTH and tMYTH baits.
• Demonstrated applicability of iMYTH for native yeast proteins with cytoplasmic C-termini.
• Demonstrated applicability of tMYTH for non-native proteins or native proteins with cytoplasmic N-termini.

Conclusions:

• Efficient MYTH bait generation is achievable using either iMYTH or tMYTH approaches.
• The choice of method depends on the specific protein's localization and origin.
• Bait generation protocols are robust and can be completed in approximately 1-2 weeks.