Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8.

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

The yeast permease Acr3p is a dual arsenite and antimonite plasma membrane transporter.

The Acr3p permease from the yeast Saccharomyces cerevisiae is a prototype member of the arsenical resistance-3 (Acr3) family of transporters, which are found in all domains of life. Remarkably little is known about substrate specificity, localization and regulation of Acr3 proteins. Here, we show that the yeast Acr3p mediates not only high-level resistance to arsenite but also moderate tolerance to antimonite. The acr3 deletion mutant shows increased sensitivity to antimonite. In addition, overexpression of the ACR3 gene complements antimonite sensitivity of cells lacking the vacuolar ABC transporter Ycf1p. Moreover, both antimonite and arsenite induce transcription of the ACR3 gene resulting in the accumulation of Acr3 transporter at the plasma membrane. However, antimonite is much weaker inducer of the ACR3 gene transcription comparing to arsenite. Interestingly, the presence of metalloids does not influence either stability of Acr3 protein or its intracellular localization suggesting that Acr3p is mainly regulated at the transcriptional level. Finally, transport experiments confirmed that Acr3p indeed mediates efflux of antimonite and thus possesses a dual arsenite and antimonite specificity.

Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p.

Saccharomyces cerevisiae uses several mechanisms for arsenic detoxification including the arsenate reductase Acr2p and the arsenite efflux protein Acr3p. ACR2 and ACR3 are transcribed in opposite directions from the same promoter and expression of these genes is regulated by the AP-1 (activator protein 1)-like transcription factor Yap8p. Yap8p has been shown to permanently associate with this promoter and to stimulate ACR2/ACR3 expression in response to arsenic. In the present study we characterized the DNA sequence that is targeted by Yap8p. We show that Yap8p binds to a pseudo-palindromic TGATTAATAATCA sequence that is related to, but distinct from, the sequence recognized by other fungal AP-1 proteins. Probing the promoter by mutational analysis, we confirm the importance of the TTAATAA core element and pin-point nucleotides that flank this element as crucial for Yap8p binding and in vivo activation of ACR3 expression. A genome-wide search for this element combined with global gene expression analysis indicates that the principal function of Yap8p is to control expression of ACR2 and ACR3. We conclude that Yap8p and other yeast AP-1 proteins require distinct DNA-binding motifs to induce gene expression and propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.