Fig 3.
Leu99, Leu102, Cys291, and Cys293 are required for the Aft1p-Grx3/4p interaction. (A) The PJ69-4A strain was transformed with an expression plasmid for AD-fused Aft1p, mutant Aft1p, or the corresponding empty vector, as well as an expression plasmid for the indicated BD-fused proteins or the corresponding empty vector. Cells were grown at 30°C for 4 days on SD medium lacking (−ADE −HIS) or containing (+ADE +HIS) adenine and histidine. Cells were spotted using 3-fold serial dilutions beginning at 600 cells per spot. (B) Lysates from the cells used for panel A were analyzed by immunoblotting with anti-HA (for the detection of AD-fused proteins) or with anti-myc (for the detection of BD-fused proteins). *, nonspecific bands. (C and D) Δgrx3 Δgrx4 cells carrying expression plasmids for Aft1-TAP or the indicated mutants and Grx3-HA (C) or Grx4-HA (D) 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. TAP-precipitates and lysates were probed with the indicated antibodies.