FIGURE 5.

Mit1, Prm1, and Mxr1 independently activate P_AOX1 in P. pastoris. A, Ni²⁺-NTA pulldown assay of protein-protein interactions of Mit1, Prm1, and Mxr1. Mit1, Prm1, and Mxr1 fused with FLAG, HA, and His₆ tags, respectively, were co-expressed in the WT strain. Cells were cultivated in methanol, and then whole cell proteins were extracted for the Ni²⁺-NTA pulldown assay. The His₆-tagged proteins were pulled down by Ni²⁺-NTA beads. The pulled-down proteins were detected by Western blotting using antibodies of their specific tags. WCE, whole cell extract; IP, immunoprecipitated proteins. + and −, indicated fusion proteins were present or absent, respectively. The interaction between C4qzn3 and Mxr1 was used as a positive control (29). Three independent replicates were performed, and the pulled-down proteins were mixed together. Twenty micrograms of total protein was loaded into each whole cell extract lane. Twenty microliters of the same pulled-down protein samples was loaded into each immunoprecipitation lane in each experiment. Normalization of signal intensity to total protein loading is described under “Miscellaneous Methods.” B, B2H assay of protein-protein interactions of Mit1, Prm1, and Mxr1. Strains with proteins that could bind with each other showed up as a blue colony on LB plates supplemented with isopropyl 1-thio-β-d-galactopyranoside and β-galactosidase. T18 and T25 are two complementary fragments of adenylate cyclase (CyaA) from Bordetella pertussis. Zip, represents the leucine zipper motif of GCN4. Strains are indicated by their expressed T18 or T25 fusion proteins. A strain expressing T25-zip and T18-zip fusion proteins was used as a positive control. It showed up as a blue colony (Cya⁺ phenotype) as the dimerization of leucine zipper motifs appended to the T25 and T18 fragments. A strain expressing T18 and T25 was used as negative control. C, BiFC assay of protein-protein interactions of Mit1, Prm1, and Mxr1. Strains expressing protein partners fused with N-terminal aa 1–173 of YFP (YN) or C-terminal aa 155–239 of YFP (YC) are indicated on each panel. The protein-protein interactions of Mit1, Prm1, and Mxr1 were tested in methanol, and the interactions of Mxr1 and C4qzn3 were tested in glucose (bottom left) and ethanol (bottom right). Positive interactions resulted in fluorescence.