Figure 2.
Maturation of the cytosolic Fe/S protein Rli1p requires the mitochondrial and cytosolic Fe/S protein assembly machineries. (A) Wild-type cells (−) or cells expressing Rli1p-HA (HA) were radiolabelled with 55Fe, cell extracts were prepared and an immunoprecipitation was performed using antibodies against the HA-tag or from pre-immune serum (PIS). The binding of 55Fe to the immunobeads was estimated by scintillation counting. (B) The assay for maturation of Rli1p-HA was performed as described in (A) using wild-type (WT), Gal-NFS1, Gal-YAH1, and Gal-ATM1 cells that were transformed with a plasmid encoding Rli1p-HA. Cells were incubated in glucose-containing, iron-poor media for the indicated time periods and an immunoprecipitation was performed using antibodies against the HA tag (upper panel). The lower panel shows the iron uptake into the cells measured by scintillation counting of cell extracts. (C) Gal-CFD1 cells transformed with a plasmid encoding Rli1p-HA were used for 55Fe incorporation into Rli1-HA after growth of cells in the presence of galactose (Gal) or glucose (Glu) as described in part B. The inset shows an immunostain of Rli1p and Cfd1p using cell extracts.