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. 2011 Oct 10;286(48):41205–41216. doi: 10.1074/jbc.M111.296152

FIGURE 3.

FIGURE 3.

The Isa proteins are essential for the maturation of aconitase-type Fe/S proteins. A, homoaconitase (Lys4) enzyme activities were determined in mitochondria isolated from WT and from depleted Gal-ISA1 or Gal-ISA2 cells overproducing Lys4-HA from vector p426-TDH3. Activities were normalized to those of malate dehydrogenase (MDH). B, isa1/2Δ cells with or without a vector overproducing Lys4p-HA were cultivated in parallel with wild-type cells (W303-1A or BY4741), lys4Δ, and aco1Δ cells for 3 days under aerobic (+O2) or anaerobic conditions (−O2) on SD minimal medium lacking either Lys or Glu. C, 55Fe binding to overproduced Lys4-HA was determined in WT, Gal-ISA1, Gal-ISA2, and Gal-ISU1/Δisu2 cells cultivated under inducing (Gal) or repressive conditions (Glc), as described in the legend to Fig. 2. Wild-type cells containing the empty plasmid (WT−) were analyzed in parallel. D, 55Fe binding to aconitase was determined in WT and the indicated Gal strains cultivated under inducing (Gal) or repressive conditions (Glc). E, the total amounts of aconitase (Aco1), Lys4-HA, porin (Por1), Isa1, Isa2, and Isu1 in extracts from the indicated cells were assessed by immunostaining. Gal-ISA strains were repressed to critical levels by cultivation in SD minimal medium for 3 days prior to analysis. Gal-ISU1/Δisu2 cells were depleted for 16 h. aco1Δ cells served as a control. Error bars, S.E. (n > 4).