Greg Keller

Role of glutaredoxin-3 and glutaredoxin-4 in the iron-regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae

The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.

Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae

The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in theS. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes inS. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated inAFT1–1 cells and attenuated in aft1null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 inMAC1 cells and the lack of expression in mac1–1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5′ promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).

Identification of FRA1 and FRA2 as Genes Involved in Regulating the Yeast Iron Regulon in Response to Decreased Mitochondrial Iron-Sulfur Cluster Synthesis

The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon.

Histidine 103 in Fra2 Is an Iron-Sulfur Cluster Ligand in the [2Fe-2S] Fra2-Grx3 Complex and Is Required for in Vivo Iron Signaling in Yeast

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S]2+-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. To further characterize this unusual Fe-S-binding complex, site-directed mutagenesis was used to identify specific residues in Fra2 that influence Fe-S cluster binding and regulation of Aft1 activity in vivo. Here, we present spectroscopic evidence that His-103 in Fra2 is an Fe-S cluster ligand in the Fra2-Grx3 complex. Replacement of this residue does not abolish Fe-S cluster binding, but it does lead to a change in cluster coordination and destabilization of the [2Fe-2S] cluster. In vivo genetic studies further confirm that Fra2 His-103 is critical for control of Aft1 activity in response to the cellular iron status. Using CD spectroscopy, we find that ∼1 mol eq of apo-Fra2 binds tightly to the [2Fe-2S] Grx3 homodimer to form the [2Fe-2S] Fra2-Grx3 heterodimer, suggesting a mechanism for formation of the [2Fe-2S] Fra2-Grx3 heterodimer in vivo. Taken together, these results demonstrate that the histidine coordination and stability of the [2Fe-2S] cluster in the Fra2-Grx3 complex are essential for iron regulation in yeast.

Independent Metalloregulation of Ace1 and Mac1 in Saccharomyces cerevisiae

ABSTRACT Ace1 and Mac1 undergo reciprocal copper metalloregulation in yeast cells. Mac1 is functional as a transcriptional activator in copper-deficient cells, whereas Ace1 is a transcriptional activator in copper-replete cells. Cells undergoing a transition from copper-deficient to copper-sufficient conditions through a switch in the growth medium show a rapid inactivation of Mac1 and a corresponding rise in Ace1 activation. Cells analyzed after the transition show a massive accumulation of cellular copper. Under these copper shock conditions we show, using two epitope-tagged variants of Mac1, that copper-mediated inhibition of Mac function is independent of induced protein turnover. The transcription activity of Mac1 is rapidly inhibited in the copper-replete cells, whereas chromatin immunoprecipitation studies showed only partial copper-induced loss of DNA binding. Thus, the initial event in copper inhibition of Mac1 function is likely copper inhibition of the transactivation activity. Copper inhibition of Mac1 in transition experiments is largely unaffected in cells overexpressing copper-binding proteins within the nucleus. Likewise, high expression of a copper-binding, non-DNA-binding Mac1 mutant is without effect on the copper activation of Ace1. Thus, metalloregulation of Ace1 and Mac1 occurs independently.

Haa1, a Protein Homologous to the Copper-regulated Transcription Factor Ace1, Is a Novel Transcriptional Activator

The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and theYRO2 homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c andYRO2 appears to be direct. Induction of HAA1using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.

Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress

The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5′-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3′. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (KD of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (KD of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (KD of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5′-(G/C)(A/C)GG(G/C)G-3′. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed.

Functional Independence of the Two Cysteine-rich Activation Domains in the Yeast Mac1 Transcription Factor

Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the264CXC(X)4CXC(X)2C(X)2H279C1 motif. The lone exception was a L260S substitution. Two additionalMAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.