FIG. 4.

Depletion of Iba57p does not result in diminished de novo Fe/S cluster formation on ferredoxin (Yah1p) or isopropylmalate dehydratase (Leu1p). (A) The indicated strains carrying p426YAH1-HA were grown in iron-poor SD medium. Cells were radiolabeled and Yah1p-HA immunoprecipitated with anti-HA immunobeads (Santa Cruz) as described in Fig. 2. Yah1p was detected by immunostaining with anti-HA. Por1p was used as a loading control. (B) S288c WT and iba57Δ cells were spotted onto minimal medium plates containing only lysine and glutamate and either containing or lacking leucine. (C) W303-1A WT, depleted Gal-IBA57, and iba57Δ cells were grown overnight in glucose minimal medium, and cell lysates were prepared by the glass bead method. The Leu1p activity was assayed immediately. (D) W303-1A (WT), Iba57p-depleted Gal-IBA57, and iba57Δ cells were grown overnight in iron-poor SD medium, and ⁵⁵Fe binding to Leu1p was analyzed by the radiolabeling and immunoprecipitation assay described in Fig. 2. Leu1p and Por1p were detected with polyclonal antiserum. (E) Indicated strains carrying pFET3-GFP were grown overnight in SD medium supplemented with 200 μM ferric ammonium citrate (iron replete) or 50 μM bathophenanthroline disulfonic acid (iron depleted), and diluted to an OD₆₀₀ of 0.2. After an additional 4 h of growth the cells were harvested and then resuspended at an OD₆₀₀ of 1, and the fluorescence emission at 513 nm was determined. Gal strains were depleted of their respective gene product by growth in SD medium.