Fig 6.

Grx3/4p require an iron-sulfur cluster to interact with Aft1p. (A and B) Grx3-TAP and Grx3-HA interact under iron-replete conditions. (A) Δgrx3 Δgrx4 cells carrying expression plasmids for Grx3-TAP and Grx3-HA were cultured in iron-free medium to mid-log-phase growth (20 h). Cells were cultured for an additional 15 min in the absence (−) or presence (+) of 200 μM FeSO₄, and TAP immunoprecipitates and cell lysates were probed with the indicated antibodies. (B) Cells expressing both Grx3-TAP and Grx4-HA (+ Aft1-TAP) or Grx4-HA alone (− Aft1-TAP) from their original chromosomal loci were cultured as for panel A, and interactions between Grx3-TAP and Grx4-HA were analyzed by immunoprecipitation. (C) Lys203, Thr251, and Cys211 of Grx3p are important for iron binding by Grx3p in vivo. Δgrx3 Δgrx4 cells carrying an expression plasmid for Grx3 (−), Grx3-TAP (WT), or the indicated mutants were cultured in iron-free medium to mid-log-phase growth. Cells were radiolabeled with 370 kBq of ⁵⁵Fe for 2 h. TAP-tagged proteins were precipitated, and bound ⁵⁵Fe was quantified by scintillation counting. Data represent mean values from three independent experiments. Error bars indicate standard deviations (SD). The amount of precipitated proteins was assessed by immunoblotting using an anti-TAP antibody. (D) Lys203, Thr251, and Cys211 of Grx3p are important for the Aft1p-Grx3p interaction. Δgrx3 Δgrx4 cells carrying expression plasmids for Aft1-TAP and Grx3-HA or the indicated mutants were cultured in iron-depleted medium to mid-log-phase growth. Cells were cultured for an additional 15 min in the presence of 200 μM FeSO₄, and TAP immunoprecipitates and cell lysates were probed with the indicated antibodies. (E) Lys203, Thr251, and Cys211 of Grx3p are important for the iron-dependent dissociation of Aft1p from the FET3 promoter. Δgrx3 Δgrx4 cells carrying expression plasmids for Aft1-TAP and Grx3p-HA or the indicated mutants were cultured in iron-free medium to mid-log-phase growth. Cells were cultured for an additional 30 min in the absence (−) or presence (+) of 200 μM FeSO₄, and Aft1p binding to the FET3 and ACT1 promoters was probed by chromatin immunoprecipitation. (F) Lys203, Thr251, and Cys211 of Grx3p are important for the regulation of FTR1 expression by iron. Δgrx3 Δgrx4 cells carrying an expression plasmid for Grx3p-HA or the indicated mutants were cultured in iron-depleted medium to mid-log-phase growth. Cells were cultured for an additional 30 min in the absence (−) or presence (+) of 200 μM FeSO₄, and the expression of FTR1, FET3, FRE1, SIT1, and ACT1 was analyzed by Northern blotting.