Fig. 1.
Principle of the MYTH 2.0 system. A, schema of the MYTH 2.0 basic principle to monitor protein-protein interactions. The MYTH 2.0 format differs from its previously described counterpart in that the reporters are placed N-terminal instead of C-terminal to the bait. The bait needs to be firmly attached to the membrane, whereas membrane anchorage of the prey is not mandatory. For a transmembrane prey protein, a default orientation of its termini is not pivotal provided that NubG resides within the cytosol. Upon association of TF-Cub-Bait with NubG-Prey, reconstitution of Ub from C- and N-terminal halves (i.e. Cub and NubG) leads to UBP-dependent proteolytic liberation (open scissors) of the transactivator TF fused to Cub and consequent activation of the reporter genes. TF is a chimera of the LexA DNA-binding domain with the VP16 activation domain. The TF triggers the induction of exogenous HIS3 and lacZ reporter genes that allow for the colorimetric detection of β-galactosidase (lacZ) and prototrophic growth without histidine (HIS3). C, C terminus; N, N terminus; PM, plasma membrane; Prey S, soluble prey; Prey M, membrane-associated prey. B, novel bait vectors (pCLB-1 and pTLB-1) for the MYTH 2.0 system. Both vectors are LEU2-based low copy number (CEN/ARS) vectors bearing either a weak yeast CYC1 (pCLB-1) or a strong TEF1 (pTLB-1) promoter. A TF (LexA-VP16) is followed by the Cub domain and the MCS. The foreign cDNA sequence encoding a transmembrane bait protein of interest is introduced into the MCS in-frame to the TF-Cub portion. Also shown in the MCS are unique PstI, Nco1, Stu1, SacII, Nco47III, AstII, and BglII restriction sites. *, stop codons.