Fig. 1. Schematic overview of the YAMTAD system.

a Schematic diagram of the strategy used for constructing the TCR and pMHC cassettes. The TCR cassette was constructed by connecting the variable domains of the TCR with a flexible linker. The pMHC cassette features an N-terminal peptide library linked to wild-type β-2-microglobulin (β2M), followed by the HLA heavy chain. TCR and pMHC cassettes are tethered to an epitope tag and Aga2p driven by the constitutive GPD promoter (pGPD). TCR and pMHC cassettes and mating-type-specific fluorescence reporters (mCherry and mTurquoise) were integrated into the ARS314 site of MATa or MATalpha yeast, respectively, using TRP1 as the selection marker. b Schematic overview of the YAMTAD system. TCR and pMHC cassettes were displayed on MATa and MATalpha yeast cell surfaces by binding to the Aga1 protein. MATa and MATalpha have complementary leucine and lysine auxotrophic markers for diploid selection. Mating is induced when a TCR displayed on MATa yeast recognizes cognate pMHC displayed on MATalpha yeast. The diploids expressing the double fluorescence can be sorted by FACS or SC-Lys-Leu screening for identification of the peptide and TCR sequences in them by next-generation sequencing.