title: Zap1 activation domain 1 and its role in controlling gene expression in response to cellular zinc status.
abstract: The Zap1 transcription factor is a central player in zinc homeostasis in yeast. This protein regulates the expression of genes involved in zinc accumulation and storage. For most of its target genes, Zap1 activates expression in zinc-limited cells and this function is inhibited in replete cells. Zap1 has two activation domains, AD1 and AD2, which are independently regulated by zinc status. In this study, we characterized AD1 and its regulation by zinc. AD1 was mapped using deletions to residues 332-402 of Zap1. The region required for the zinc responsiveness of this activation domain, designated 'ZRD(AD1), was mapped to residues 182-502. Thus, AD1 is embedded within its larger zinc-responsive domain. Using a combination of in silico analysis, random mutagenesis and site-directed mutagenesis, we identified key residues within ZRD(AD1) required for its regulation by zinc. Most of these residues are cysteines and histidines that could potentially serve as Zn(II) ligands. These results suggest that ZRD(AD1) senses zinc by direct Zn(II) binding. Consistent with this hypothesis, purified ZRD(AD1) bound multiple Zn(II) ions. Finally, our results indicate that, in the context of the full-length Zap1 protein, AD1 and AD2 are both critical to the full control of gene expression in response to zinc.
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Herbig
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Bird
authors.forename: Sabina
authors.initials: S
authors.lastname: Swierczek
authors.forename: Keith
authors.initials: K
authors.lastname: McCall
authors.forename: Michelle
authors.initials: M
authors.lastname: Mooney
authors.forename: Chang-Yi
authors.initials: CY
authors.lastname: Wu
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Winge
authors.forename: David J
authors.initials: DJ
authors.lastname: Eide
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C103264:ZAP1 protein, S cerevisiae; D015032:Zinc