Figure 8. Overexpressing Zap1 disrupts gene regulation and zinc homeostasis.

A) ZHY6 zap1Δ cells transformed with pGEV, the pGI-1 ZRT1-lacZ reporter and either pZap1^WT or pYef2 vector were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂. To induce high Zap1 expression, cells were treated with 1 µM β-estradiol (ox). Cells were then harvested and β-galactosidase assays were performed. B) Wild-type DY1457 cells transformed with pGEV, the pGI-1 ZRT1-lacZ reporter, and either pYef2 or pZap1^mZnf4 were grown to exponential phase in LZM supplemented with the indicated concentration of ZnCl₂. To induce high Zap1^mZnf4 levels, cells were treated with 1 µM β-estradiol (ox). Cells were then harvested and β-galactosidase assays were performed. The values shown in panels A and B are the means of three independent cultures, and the error bars equal ±1 S.D. C) Protein extracts were generated from the samples described in panels A and B and subjected to immunoblot analysis using antibodies against Zap1 (myc) and Pgk1. D) Wild-type DY1457 cells and ZHY6 zap1Δ cells transformed with pGEV and either the pYef2 vector or pZap1^WT were inoculated at an A₆₀₀ of 0.02 in LZM supplemented with the indicated concentration of ZnCl₂ plus tracer amounts of ⁶⁵ZnCl₂. To induce high levels of Zap1^WT protein expression, cultures were treated with 1 µM β-estradiol (Zap1^WTox). Cultures were grown to an A₆₀₀ of ∼0.75, after which zinc accumulation was measured. Shown are the means of three independent cultures and the error bars indicate ±1 S.D. The asterisks indicate a significant difference (p<0.05) of zap1Δ cells expressing Zap1^WTox relative to Zap1^WT controls as determined by 2-sided Student t-test.