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Fig. 4. Analysis of Yap8p cysteine mutants. (A) Cartoon of Yap8p with the bZIP DNA-binding domain and cysteine residues indicated. (B) Phenotypes of Yap8p cysteine to alanine mutations. Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into W303-1A yap8
. Transformants were grown in liquid medium and tenfold serial dilutions of the cultures were spotted on agar plates containing sodium arsenite. Growth was scored after 2 days at 30°C. (C) 
-galactosidase assays. The W303-1A yap8 mutant 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); 6 hours). The induction level of ACR3-lacZ in the presence of wild-type Yap8p-HA was set to 100 and the ACR3-lacZ induction levels in the presence of the Yap8p mutants are given relative to that of wild-type Yap8p-HA. Error bars represent ± s.d. (D) Yap8p-C132A, C137A and C274A fail to stabilize in the presence of As(III). Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into W303-1A yap8 and Yap8p-HA protein levels were monitored by western blot analysis prior to and 1 hour after addition of 0.2 mM As(III).
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