Figure 3.

Depletion of Yah1p results in a defect in the assembly of an Fe/S cluster into a mitochondrial apoprotein. (A) Mitochondria (M) and postmitochondrial supernatants (PMS) of wild-type (WT) and ΔLWT-ML cells expressing a mitochondrial version of Leu1p were subjected to SDS/PAGE and immunostaining with antisera against Leu1p and matrix-exposed Tim44p. (B) ΔLWT-ML and ΔLGY-ML cells expressing Leu1p in mitochondria were grown in iron-free minimal medium containing glucose for the indicated times. Cells were radiolabeled with (⁵⁵Fe) iron chloride in the presence of 1 mM ascorbate for 1 h; cell lysates were prepared; and the uptake of ⁵⁵Fe was quantitated by liquid scintillation counting (Lower). Aliquots of the lysates were subjected to SDS/PAGE and immunostaining with antisera directed against Leu1p (α-Leu1p stain; Bottom Insert). Immunoprecipitation with anti-Leu1p antibodies was performed, and coprecipitated ⁵⁵Fe was estimated by liquid scintillation counting (Upper; ref. 7). The background signal obtained for immunoprecipitation performed with preimmune serum (usually less than 2 × 10³ cpm per g of cells) was subtracted.