Overexpressing Zap1 disrupts gene regulation and zinc homeostasis.

<p><b>A</b>) ZHY6 <i>zap1Δ</i> cells transformed with pGEV, the pGI-1 <i>ZRT1-lacZ</i> 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>B</b>) Wild-type DY1457 cells transformed with pGEV, the pGI-1 <i>ZRT1-lacZ</i> 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. <b>C</b>) 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. <b>D</b>) Wild-type DY1457 cells and ZHY6 <i>zap1Δ</i> 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 <i>asterisks</i> indicate a significant difference (<i>p</i><0.05) of <i>zap1Δ</i> cells expressing Zap1^WTox relative to Zap1^WT controls as determined by 2-sided Student <i>t</i>-test.