Yeast Two-Hybrid Assay for Pex5 Interactions

Sequences encoding the TPR domains of Pex5a and Pex5b were inserted into pGBKT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech) between the EcoRI and BamHI restriction sites via Gibson assembly. The ORFs for GFP or GFP modified with C-terminal dodecamers of Um01966, Um10665, Um11001, and Um03158 including PTS1 motifs were cloned into pGADT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech) between the EcoRI and BamHI restriction sites. Either pGBKT7-Pex5aTPR or pGBKT7-Pex5bTPR were co-transformed with one of the pGADT7 plasmids into YTS398, a derivative AH109 of deleted for pex5 (Stehlik et al., 2020 (link)). Three independent transformants of each of the 10 combinations were grown in liquid synthetic defined (SD) medium lacking leucine and tryptophan to an OD₆₀₀ of approx. 1. Cells were washed once with sterile water and 4 µl of fivefold or fiftyfold dilutions (OD₆₀₀ = 0.2 or 0.02) were spotted on solid SD medium lacking leucine and tryptophan as growth control, and on SD medium lacking leucine, tryptophan and histidine, and containing 1.5 mM 3-amino-1,2,4-triazole to test for protein – protein interaction. Plates were incubated for 3 days at 30°C.