Fig. 5. Yap8p functions as a homodimer in vivo. (A) Phenotypes of wild-type W303-1A cells expressing Yap8p-HA cysteine to alanine mutants. Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into wild-type cells and growth of the transformants was scored as above. (B) -galactosidase assays. The W303-1A wild type was co-transformed with a plasmid containing the indicated version of Yap8p-HA and a plasmid containing the ACR3-promoter-lacZ fusion gene. Transformants were assayed for -galactosidase activity as described in Materials and Methods. ACR3-lacZ induction levels were calculated by comparing -galactosidase activities in untreated and As(III)-exposed cells (0.1 mM As(III); 4 hours). The induction level of ACR3-lacZ in the wild-type strain with the empty vector was set to 100 and the ACR3-lacZ induction levels in the presence of the indicated Yap8p versions are given relative to that of wild-type cells with the empty vector. Error bars represent ± s.d. (C) Yap8p forms homodimers in vivo. Co-IP assays were performed with cells co-transformed with two epitope-tagged versions of Yap8p (Myc₉-Yap8p and Yap8p-HA). Myc₉-Yap8p was immunoprecipitated with an anti-Myc antibody, and the presence of wild-type or mutant forms of Yap8p-HA in the precipitates was detected using an anti-HA antibody. Cells were either untreated or exposed to 0.2 mM As(III) for 1 hour as indicated. The upper panel shows expression levels of wild-type and mutant forms of Yap8p-HA in cells coexpressing Myc₉-Yap8p (Input) whereas the lower panel shows Co-IP. Proteins were separated by SDS-PAGE and the presence of Yap8p was monitored with anti-Myc and anti-HA antibodies.