Fig. 1. Regulation of Zap1 activity by zinc occurs at a post-translational level. (A) Wild-type (DY1457) and zap1 mutant (ZHY6) cells containing either the pYef2 vector or pMyc-Zap11–880 were grown to exponential phase in LZM-galactose supplemented with either 5 µM (–Zn) or 1000 µM (+Zn) ZnCl2. Zap1 activity in each strain was assessed using the pDg2 ZRE-lacZ reporter. ZHY6 pMyc-Zap11–880 transformants were also assayed after growth in glucose, a carbon source that represses most but not all expression from the GAL1 promoter. A representative experiment is shown and the error bars indicate 1 SD. (B) The stability of Zap1 is not affected by zinc status. Wild-type (DY1457) cells transformed with the pYef2 vector and zap1 mutant (ZHY6) cells bearing pMyc-Zap11–880 were grown in LZM-galactose to exponential phase. The concentrations of ZnCl2 added to the medium were 5 (lane 2), 250 (lane 3), 500 (lane 4) and 1000 µM (lanes 1 and 5). Crude protein extracts were prepared, fractionated by SDS–PAGE analysis, and assayed for Zap1 and Vph1 protein levels by immunoblotting.