FIGURE 8.

Loss of Fe-S cluster synthesis does not lead to decreased Yap5 levels. A, cells (wild type, Δyap5, Δccc1, Δccc1Δisu1, Δssq1Δccc1 (pMET3SSQ1), and Δyfh1Δccc1(pMET3YFH1)) were grown in CM medium in the presence (off) or absence (on) of methionine overnight. Cells were harvested, treated with 10% trichloroacetic acid, and broken with glass beads, and cell extracts were analyzed by Western blot using a polyclonal rabbit anti-Yap5 and a mouse anti-carboxypeptidase Y (CPY) antibody. The arrow denotes the Yap5-specific band. A representative blot is shown. Western blots were quantified using carboxypeptidase Y as a loading control from three separate experiments. B, cells (Δssq1Δccc1 (pMET3SSQ1)) containing a CCC1-lacZ reporter construct were transformed with a high copy plasmid containing HIS-tagged Yap5 under the control of its endogenous promoter. Cells were grown as in A, and HIS-Yap5 levels were determined by Western blot using mouse anti-HIS antibody. CCC1-lacZ activity in these cells was determined. The data are presented as specific activity, and the error bars reflect the S.D. of three independent experiments.