A second iron-regulatory system in yeast independent of Aft1p

Abstract

Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Deltaaft2Delta deletion was generated. Growth assays established that the single aft2Delta strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Deltaaft2Delta strain was more sensitive to low-iron growth conditions than the single-mutant aft1Delta strain. A mutant allele of AFT2 (AFT2-1(up)), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Delta strain. The AFT2-1(up) allele also increased the uptake of (59)Fe in an aft1Delta strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1(up)-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1(up) allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.

Figure 1

Sequence comparison of Aft1p and Aft2p. The amino acid sequences of Aft1p and Aft2p of S. cerevisiae were aligned by using the clustalw program. The conserved CXC motif is underlined and in bold (see text).

Figure 2

AFT2 partially complements the aft1Δ mutant strain. Overnight cultures of each strain were grown in CM(−Ura) medium containing glucose. The cultures were washed, and 10-fold serial dilutions were spotted onto CM(−Ura) agar plates containing either glucose or glycerol, which were then incubated at 30°C.

Figure 3

The effect of iron on the growth of the aft mutant strains. Overnight cultures of the wild-type strain and each of the aft-mutant strains were grown in liquid CM(−Ura) with glucose medium. Each strain was grown in duplicate, with one of each pair being supplemented with FeCl₂ (100 μM). Iron supplementation in the pregrowth medium is indicated by “+ Fe.” The cells were washed, and 10-fold serial dilutions were spotted onto (A) complex medium (YPD) agar plates with or without supplemented FeCl₂ (100 μM) or (B) synthetic medium (CMD) agar plates with or without supplemental FeCl₂ (100 μM) or BPS (10 μM). Plates were incubated at 30°C and, in the case of one YPD plate, under anaerobic conditions.

Figure 4

S1 nuclease protection assays to quantify mRNA levels of genes activated by the AFT2-1^up allele. RNA was isolated from (A) the aft2Δ strain and (B) the aft1Δ strain transformed with pRS416 (−), pAFT2 (+), and pAFT2-1^up (up) and grown in CM(−Ura) with glucose medium to mid-log phase. The upper band for each sample is the specified gene, and the lower band the calmodulin loading control (CMD1).

Figure 5

Aft2p is a direct transcriptional activator that responds to low iron. S1 nuclease protection assays were carried out by using RNA isolated from (A) the aft2Δ strain transformed with either pGAL-AFT2-1^up or pYeF2 (control), which were grown in CM(−Ura) with raffinose to mid-log phase, at which time 2% galactose was added to induce the transcription of the AFT2-1^up allele. Cells were harvested at the specified times (B), the aft1Δaft2Δ strain was transformed with pRS416 (control), pAFT2, and pAFT2-1^up, which were pregrown overnight with 10 μM FeCl₂, harvested, washed, and resuspended in CMD(−Ura) medium with or without supplemental FeCl₂ (100 μM) or BPS (100 μM) and then grown exponentially for 8 h. The Upper band for each sample is the specified gene, and the Lower band the calmodulin loading control (CMD1).

Figure 6

Aft1p and Aft2p bind to the same promoter fragment in vitro but differentially regulate gene expression. (A) S1 nuclease protection assays to quantify mRNA levels of genes activated by the AFT1-1^up and AFT2-1^up alleles. RNA was isolated from the aft1Δaft2Δ strain transformed with a control plasmid (−) (pRS316 in the case of pAFT1-1^up and pRS416 in the case of pAFT2-1^up), pAFT1-1^up (1^up), and pAFT2-1^up (2^up) and grown in CMD(−Ura) medium to mid-log phase. The Upper band for each sample is the specified gene, and the Lower band the calmodulin loading control (CMD1). (B) Gel retardation assays were performed by using a ³²P-labeled 30-bp duplex of the FET3 promoter as probe with no protein (lane 1), and lysates from E. coli containing pET3 (lane 2), pAft1-313 (lane 3), and pAft2-214 (lane 4).

Figure 7

Iron uptake in the aft mutants. The level of ⁵⁹Fe uptake was analyzed in the following cultures. (A) The wild-type strain and the aft-deletion strains grown to mid-log phase in synthetic medium (CMD) with BPS (100 μM). (B) The wild-type strain and the aft1Δ strain containing either pRS416 (WT, 1Δ) or pAFT2-1^up (WT/^up, 1Δ/^up) grown to mid-log phase in CMD(−Ura) medium.