Figure 4
Molecular basis of the Yap5-Hap5 interaction. (A) Multiple alignments of the Hap4L domains of Hap4 (from S. cerevisiae), wild type C. glabrata Yap5, Yap5-Hap4LΔ and Yap5-mut2. For the latter, the substitutions are highlighted in red. (B) ChIP-QPCR was performed on strains expressing a myc-tagged Yap5 in presence (wild type) or absence (hap5Δ) of HAP5 and on strains expressing the two different Yap5 mutant versions. All strains were grown in YPD. The values represent the IP/Input ratios of the GRX4 promoter relative to the enrichment of the YHB1 promoter (used as an internal control), expressed as a percentage of the enrichment obtained for the wild type Yap5. The experiments were performed twice on biologically independent samples. Error bars hence represent the standard error of the mean. (C) Western blot analyses of the co-immunoprecipitation experiments using Hap5-Protein A as bait and wild type or mutated versions of Yap5-myc as prey. Upper panel: input samples (INPUT), lower panel: immunoprecipitated samples (IP). Immunoblotting was performed with a mouse anti-myc antibody (Roche). The Yap5 protein is fused to 13 c-Myc epitopes and the corresponding band is expected at 65 kDa. The Hap5-Protein A fusion is expected at 45 kDa and is also detected by the anti-myc antibody (although with a low affinity), because Protein A non-specifically interacts with IgG. Note the similar intensity of the Hap5-ProtA bands in the IP, which indicates that the IP efficiency was equivalent from one lane to another. The star indicates the 50 kDa band corresponding to the large chain of the anti-Mouse antibodies used for the IP. The co-immunoprecipitation experiment was performed twice on biologically independent samples and gave consistent results. The ladder on the right was copied and pasted from the white light image of the membrane. Immunoblotting of the same membranes with rabbit IgG-HRP polyclonal antibody (PAP; code Z0113; Dako), which has a high affinity for Protein A, can be found in Supplementary File S7.
