FIGURE 5.
Met4 protein level is regulated post-transcriptionally by zinc and by Zap1. A, immunoblot analysis of Met4 protein levels in wild type cells grown in LZM supplemented with a range of added zinc (lanes 2–6). A met4Δ strain (TAL31) was used as a control (lane 1). In addition, wild type and zap1Δ mutant cells (ZHY6) were grown in low zinc (−Zn, LZM + 1 μm ZnCl2) (lanes 7 and 8) and wild type cells with and without expressing the constitutive Zap1TC allele were grown in zinc-replete SD medium (+Zn, lanes 9 and 10). The multiple Met4 bands represent differences in ubiquitination and/or phosphorylation states of the protein. 3-Phosphoglycerate kinase (Pgk1) protein was used as a loading control. B, cells were grown under the same conditions as in A. RNA was isolated and was then analyzed by S1 nuclease protection assay. No change in MET4 mRNA was detected indicating the changes in protein level are because of post-transcriptional effects.