title: The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation.
abstract: The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S](2+) cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mossbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
authors.forename: Haoran
authors.initials: H
authors.lastname: Li
authors.forename: Daphne T
authors.initials: DT
authors.lastname: Mapolelo
authors.forename: Nin N
authors.initials: NN
authors.lastname: Dingra
authors.forename: Sunil G
authors.initials: SG
authors.lastname: Naik
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Lees
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Riggs-Gelasco
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.forename: Caryn E
authors.initials: CE
authors.lastname: Outten
mesh_terms: D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004795:Enzyme Stability / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D054477:Glutaredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: C529266:FRA2 protein, S cerevisiae; D054477:Glutaredoxins; C516012:Grx4 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; C545181:Grx3 protein, S cerevisiae; D010088:Oxidoreductases; D003545:Cysteine