Zap1 activation domain 1 and its role in controlling gene expression in response to cellular zinc status

Abstract

SummaryThe 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.

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Both ADs have been highly conserved in the Hemiascomycetes, indicating that both are important for Zap1 function. Mutation of potential Zn +2 ligands in AD1 blocks the inhibition of Zap1 activity that normally occurs in response to high Zn +2 levels (Herbig et al 2005). Purified AD1 binds multiple Zn +2 atoms, consistent with a direct role for metal binding.…”

“…The DNA-binding domain consists of C2H2 zinc fingers 3-7 (black boxes). AD1 is within the zincresponsive domain (ZRD) that is Cys and His rich (Herbig et al 2005). AD2 is within finger 2 but both fingers 1 and 2 are required for zinc responsiveness (Bird et al 2000).…”

The Double Zinc Finger Domain and Adjacent Accessory Domain from the Transcription Factor Loss of Zinc Sensing 1 (Loz1) Are Necessary for DNA Binding and Zinc Sensing

“…These include C 2 H 2 -type zinc fingers in Zap1, MTF-1, and now Loz1 and a cysteine/histidinerich domain in AD1 from Zap1 (4,22). Deletion of a cysteinerich metallothionein-like domain from the copper-responsive regulator Crr1 in Chlamydomonas also leads to increased expression of genes required for zinc uptake and the hyperaccumulation of zinc, suggesting that this may be another type of zinc-sensing domain (25,26). Despite a growing number of eukaryotic zinc-responsive factors, it is largely unknown whether these factors sense changes in zinc levels through zinc ion binding or if they are indirectly regulated by zinc (e.g.…”

The Cation Diffusion Facilitator Gene cdf-2 Mediates Zinc Metabolism in Caenorhabditis elegans

“…Reflecting its multiple uses, organisms that do not obtain adequate dietary zinc display a wide range of defects (Cole et al 1999;Kambe et al 2004;Chowanadisai et al 2005;Herbig et al 2005;Dufner-Beattie et al 2007). For example, humans that are zinc deficient have abnormalities of multiple systems including the skin and immune system (Hambidge 2000;Chowanadisai et al 2006;Hambidge and Krebs 2007;Maverakis et al 2007).…”