Etp1 confers arsenite resistance by affecting ACR3 expression

Abstract

In a high-throughput yeast two-hybrid screen of predicted coiled-coil motif interactions in the Saccharomyces cerevisiae proteome, the protein Etp1 was found to interact with the yeast AP-1-like transcription factors Yap8, Yap1 and Yap6. Yap8 plays a crucial role during arsenic stress since it regulates expression of the resistance genes ACR2 and ACR3. The function of Etp1 is not well understood but the protein has been implicated in transcription and protein turnover during ethanol stress, and the etp1∆ mutant is sensitive to ethanol. In this current study, we investigated whether Etp1 is implicated in Yap8-dependent functions. We show that Etp1 is required for optimal growth in the presence of trivalent arsenite and for optimal expression of the arsenite export protein encoded by ACR3. Since Yap8 is the only known transcription factor that regulates ACR3 expression, we investigated whether Etp1 regulates Yap8. Yap8 ubiquitination, stability, nuclear localization and ACR3 promoter association were unaffected in etp1∆ cells, indicating that Etp1 affects ACR3 expression independently of Yap8. Thus, Etp1 impacts gene expression under arsenic and other stress conditions but the mechanistic details remain to be elucidated.

Keywords: Acr3; Etp1; Yap8; arsenic; metalloid.

Figure 1.

The etp1Δ mutant is sensitive to As(III). (A) Ten-fold serial dilutions of the indicated strains were plated onto YNB agar plates with or without As(III) and As(V) at the indicated concentrations. Growth was monitored after 2–3 days at 30°C. Growth assays were performed with at least two biological replicates and a representative image is shown. (B)YAP8 overexpression improves As(III) resistance in the etp1Δ mutant. An empty plasmid or a plasmid carrying GFP-tagged YAP8 (endogenous YAP8 promoter, episomal plasmid) was transformed into the indicated strains and growth assays performed as above. (C)ACR3 overexpression improves As(III) resistance in the etp1Δ mutant. Growth assays were performed as above with the indicated strains transformed with an empty plasmid or a plasmid carrying ACR3 behind its endogenous promoter on a multicopy plasmid.

Figure 2.

ACR3 expression is lower in As(III)-exposed etp1Δ cells. (A) β-Galactosidase activity driven by the ACR3-promoter-lacZ fusion construct was measured in the indicated strains. Cells were exposed to 0.25 mM As(III) for 6 h or left untreated for the control. The values are the means of three biological replicates performed in triplicate ± standard deviation (SD). *P < 0.05 and **P < 0.005. (B) Cells were exposed to 0.25 mM As(III) and samples for RNA extraction were taken at the indicated time points followed by qPCR as described in the 'Materials and Methods' section. ACR3 expression was normalized to IPP1 expression. Values are the means of three biological replicates ± SD. *P < 0.05 and **P < 0.005.

Figure 3.

Etp1 does not regulate Yap8 ubiquitination and stability. (A) Yap8 stability is unaffected in etp1Δ cells. Yap8-HA was expressed from the constitutive TPI1 promoter in the wild type and the etp1Δ mutant and samples were taken for SDS–PAGE at the indicated time points. Cells were exposed to 0.5 mM As(III) for 1 h, then washed and placed in growth medium with or without As(III) as indicated. Cycloheximide (50 µg/ml CHX) was added as indicated to inhibit de novo protein synthesis. Immunoblotting was performed with anti-HA and anti-Pgk1 as loading control. The assay was performed with at least two biological replicates and a representative image is shown. (B) Yap8 ubiquitination is unaffected in etp1Δ cells. Ubiquitination of Yap8-HA was monitored in the indicated cells expressing 10× histidine (His)-tagged ubiquitin. Total cell extracts and ubiquitin conjugates eluted after immobilized-nickel affinity chromatography were separated by SDS–PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. The ubiquitin pulldown assay was performed with at least two biological replicates and a representative image is shown.

Figure 4.

Etp1 does not regulate Yap8 nuclear localization and ACR3 promoter association. (A) Nuclear localization of Yap8 is unaffected in etp1Δ cells. Yeast chromatin was fractionated as described in the 'Materials and Methods' section and the presence of Yap8-HA was monitored by immunoblotting. T, total extract; C, cytosolic fraction; Ch, chromatin fraction. Immunoblotting was performed with antibodies against the HA-tag, histone H2A as a marker for the chromatin fraction and glucose-6-phosphate dehydrogenase (G6PDH) as a marker for the cytosolic fraction. The assay was performed with at least two biological replicates and a representative image is shown. (B)In vivo occupancy of Yap8 on the ACR3 promoter is unaffected in etp1Δ cells as determined by ChIP. The indicated cells carrying Yap8-HA or the empty vector were either untreated (control) or exposed to 0.5 mM As(III) for 30 min, and qPCR was performed on chromatin fragments isolated after immunoprecipitation using an anti-HA antibody as described in the 'Materials and Methods' section. Data from three biological replicates are shown, and the error bars represent standard deviations.