Fig. S4

A Putative Yeast Homolog of Bacterial EntA Has Functional Similarities to Mammalian BDH2, Related to [Figure 4] and [Figure 7]

(A) PCR analysis confirming the deletion of the yeast YIR035C gene and replacement with a KanMX gene cassette. The positions of the PCR primer-pairs are indicated in the schematic diagram, below.

(B) Growth of wild-type and yir035cΔ strains. (Top) Five-fold serial dilutions were plated onto CSM dextrose in the absence or presence of the iron (Fe²⁺) chelator ferrozine (1 mM), and plates were incubated at 30°C for 1 day. (Bottom) Serial dilutions were plated onto CSM dextrose containing ferrozine (1 mM) in the absence or presence of 250 μM FeSO₄. The results show that in medium containing low iron a yeast strain lacking YIR035C displays a growth defect, which is alleviated by addition of iron to the medium.

(C) Control experiments showing the growth of two yeast strains deficient in genes that have well-characterized roles in iron utilization, FET3 and MRS3/4. Five-fold serial dilutions were plated onto CSM dextrose, or CSM dextrose containing ferrozine (1 mM) in the absence or presence of 250 μM FeSO₄. The results show that the fet3Δ and mrs3/4Δ strains have an iron-dependent growth defect similar to that of the yir035cΔ strain.

(D) Cytosolic aconitase activity was monitored in wild-type or yir035cΔ strains grown in CSM dextrose or CSM dextrose containing ferrozine (1 mM). The results show that the yir035cΔ strain has reduced cytoplasmic aconitase activity, which is greatly exacerbated in medium containing low iron.

(E) Heme synthesis was monitored in wild-type or yir035cΔ strains grown in CSM glycerol. The results show that heme synthesis in the yir035cΔ strain is markedly reduced.