Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

Abstract

cThe AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.A rsenic is a highly toxic metalloid that is widespread in the environment. Consequently, nearly all organisms have developed sophisticated strategies for arsenic detoxification and tolerance acquisition (1). These mechanisms include increased export from cells, sequestration in organelles, chelation by metal-binding proteins and peptides, and diminished uptake. Many detoxification systems are controlled at the transcriptional level by regulatory proteins that are activated during arsenic exposure (2-5). The best-studied arsenic-responsive regulator is the Escherichia coli ArsR repressor protein (6). In untreated cells, ArsR is bound to DNA and represses transcription of arsenic tolerance genes. Upon binding of trivalent arsenite to vicinal cysteine residues, ArsR dissociates from the promoter, resulting in induced gene expression (7). ArsR-type repressors are widespread in bacteria and archaea but are absent in higher eukaryotes. In fact, very little is known about arsenic-sensing regulators in eukaryotes.Global gene expression analyses and gene-specific studies have implicated several proteins that might mediate the transcriptional response to arsenic in the eukaryotic microbe Saccharomyces cerevisiae (budding yeast) (2,8). Two of these transcriptional regulators are Yap1 and Yap8 (also called Acr1 and Arr1). Both proteins belong to the yeast AP-1 family of transcription factors and contain conserved basic leucine-zipper (bZIP) DNA binding domains and conserved cysteine residues (9). Yap1 controls expression of genes encoding functions in oxidative stress defense and sulfur metabolism in response to arsenite exposure (10-12). Yap8 has a highly specialized function and regulates expression of only two genes: ACR2, encoding an arsenate [As(V)] reductase, and ACR3, encoding an arsenite [As(I...

A Network of Paralogous Stress Response Transcription Factors in the Human Pathogen Candida glabrata

“…ScYap5 is responsible for the activation of the high iron stress response (Li et al, 2008 ; Pimentel et al, 2012 ). ScYap7, the ohnolog of ScYap5, was recently found to be a repressor of the nitric oxide oxidase encoding gene YHB1 (Merhej et al, 2015 ) and ScYap8 is involved in the response to arsenic (Bobrowicz et al, 1997 ; Amaral et al, 2013 ; Kumar et al, 2015 ).…”

The ancillary N-terminal region of the yeast AP-1 transcription factor Yap8 contributes to its DNA binding specificity

“…Moreover, we confirmed that Arg7, Gly10 and Arg11 facilitate Yap8 high affinity binding to the Y8RE motif ( Figure 5C Our data show that Asn20 adjacent to the basic region is critical for high affinity binding of Yap8 to the 13 bp long Y8RE motif. The model suggests that Asn20 is not in direct contact with DNA, but influences the conformational space of the N-terminal tails (region [9][10][11][12][13][14][15][16][17][18][19]. The MD simulations of N20A Yap8 mutant-DNA complex showed that the mutant exhibits less stable contacts between the N-terminal and DNA, which could influence the overall stability of Yap8-DNA complexation.…”

“…The crystal structure of the Pap1 bZIP domain bound to the 8 bp YRE revealed that five amino acid residues of the basic region make direct contacts with a TTAC half-site, and these residues constitute the signature DNA recognition NxxAQxxFR sequence (2). This motif is highly conserved among members of the Pap1 subfamily suggesting that Yap proteins share a common mechanism of DNA binding (Figure 1 Yap8 sense the presence of these agents and coordinate activation of gene expression required for alleviation of metalloid toxicity (7)(8)(9)(10). Yap1 stimulates transcription of a large set of genes encoding proteins that are involved in adaptation to arsenic-induced oxidative stress and metalloid detoxification (7,9,11,12).…”

AP-1 (bZIP) Transcription Factors as Potential Regulators of Metallothionein Gene Expression in Tetrahymena thermophila

“…Besides TATA boxes and transcriptional start regions, MT gene promoters can have different motifs related to their expression induction, such as; metal responsive elements (MREs), antioxidant responsive elements (AREs), and glucocorticoid responsive elements (GREs) ( Gunther et al, 2012 ). Moreover, other binding-sites have been detected in some MT gene promoters, as GATA factors binding-sites ([A/T]GATA[A/G]) described in MTs from the nematode Caenorhabditis elegans ( Moilanen et al, 1999 ), AP-1 ( A ctivating P rotein-1 ) sites (TGA[G/C]TCA) and the GC boxes (GGGGCGGGG) related to the binding of bZIP ( b asic region leucine zip per ) TFs ( Shaulian and Karin, 2002 ; Haq et al, 2003 ; Jenny et al, 2006 ; Gutierrez et al, 2011 ; Kumar et al, 2015 ). A MTCM1 motif ( M e t allothionein C onserved M otif 1 ) has been described in the promoters of several MT genes from the ciliate Tetrahymena thermophila : MTT1 gene promoter has 6 MTCM1 motifs, MTT3 only has 2 motifs and MTT5 has 13 MTCM1 motifs in its duplicated promoter region ( Diaz et al, 2007 ; Gutierrez et al, 2011 ; de Francisco et al, 2016 ).…”

“…They are also involved in gene expression regulation processes in response to different stimulus: cytokines, growth factors, viral or bacterial infections and stress conditions ( Durchdewald et al, 2009 ). Likewise, AP-1 has been related to MT gene expression increasing levels under different stress conditions in different organisms, such as; Saccharomyces cerevisiae ( Wu et al, 1993 ; Kumar et al, 2015 ), Crassostrea virginica ( Jenny et al, 2006 ), Drosophila melanogaster ( Kockel et al, 2001 ), and C. elegans ( Moilanen et al, 1999 ). An increase of MT-1 metallothionein gene expression level has been related to a higher gene expression of a gene encoding an AP-1 TF in mammalian cells after treatment with an oxidative stressor ( Braithwaite et al, 2010 ).…”