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First published online 2 January 2007
doi: 10.1242/jcs.03346

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Regulation of the arsenic-responsive transcription factor Yap8p involves the ubiquitin-proteasome pathway

Department of Cell and Molecular Biology/Microbiology, Göteborg University, S-405 30 Göteborg, Sweden

View larger version (27K): [in this window] [in a new window]   Fig. 1. Yap8p is stabilized in the presence of arsenite. (A) Yap8p-HA was expressed from the constitutive TPI1 promoter in the yap8 mutant (W303-1A background) and samples were taken for western blot analysis at the time-points indicated prior to and after exposing cells to increasing concentrations of sodium arsenite [As(III)]. The concentrations used are indicated above the blot. Hog1p was used as loading control and Yap8p protein levels were quantified and normalized to the Hog1p level of each lane. (B) Cells expressing Yap8p-HA were exposed to 0.5 mM As(III) for 1 hour (panel A), then washed and placed in growth medium with or without As(III) (panels B-E). Cycloheximide (0.1 mg/ml) was added as indicated and Yap8p-HA levels were monitored by western blot analysis. Yap8p protein levels in the presence of cycloheximide (CHX) were quantified and normalized to the Hog1p level as described above. (C) Yap8p-HA levels correlate with the cytosolic As(III) concentration. Yap8p protein levels were quantified as described above in wild-type, acr3, ycf1 and acr3 ycf1 cells treated with 10 µM As(III) for the indicated time.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Yap8p degradation involves the ubiquitin-proteasome pathway. (A) The erg6 mutant (BY4741 background) was transformed with a plasmid expressing Yap8p-HA. Proteasome activity was reduced by adding MG132 (0.1 mM) to cell cultures and Yap8p-HA levels were monitored as above. Since MG132 was dissolved in DMSO, an equal volume of this solvent was added to the control sample. A long exposure time was needed to visualize Yap8p-HA in the control sample of the erg6 strain. (B) Yap8p levels increase in the pre1-1 pre4-1 mutant that is defective in the -type subunits of the catalytic 20S core of the proteasome. The Yap8p-HA plasmid was transformed into wild type (WCG4a background) and the pre1-1 pre4-1 mutant, and Yap8p-HA levels were monitored by western blot analysis. (C) The rate of Yap8p degradation is reduced in cells lacking the ubiquitin-conjugating enzyme Ubc4p. Cells (in BY4741 background) expressing Yap8p-HA were exposed to 0.5 mM As(III) for 1 hour, then washed and placed in growth medium without As(III). Cycloheximide (0.1 mg/ml) was added and Yap8p-HA levels were monitored by western blot analysis. Yap8p protein levels were quantified and normalized to the Hog1p level of each lane.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Yap8p stabilization contributes to increased transcriptional activity. An ACR3-promoter-lacZ fusion construct was transformed into wild-type cells and into ubiquitin-proteasome pathway mutants. Transformants were assayed for -galactosidase activity as described in Materials and Methods. The results are the average of three independent experiments and the error bars represent s.d. (A) -galactosidase activity measurements in the pre1-1 pre4-1 mutant (WCG4a background). (B) -galactosidase activity measurements in ubc4 and ubc5 mutants (DF5 background). (C) -galactosidase activity measurements in wild-type cells (WCG4a background) transformed with an empty vector or with a plasmid carrying Yap8p-HA.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Analysis of Yap8p cysteine mutants. (A) Cartoon of Yap8p with the bZIP DNA-binding domain and cysteine residues indicated. (B) Phenotypes of Yap8p cysteine to alanine mutations. Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into W303-1A yap8. Transformants were grown in liquid medium and tenfold serial dilutions of the cultures were spotted on agar plates containing sodium arsenite. Growth was scored after 2 days at 30°C. (C) -galactosidase assays. The W303-1A yap8 mutant was co-transformed with a plasmid containing the indicated version of Yap8p-HA and a plasmid containing the ACR3-promoter-lacZ fusion gene. Transformants were assayed for -galactosidase activity as described in Materials and Methods. ACR3-lacZ induction levels were calculated by comparing -galactosidase activities in untreated and As(III)-exposed cells (0.1 mM As(III); 6 hours). The induction level of ACR3-lacZ in the presence of wild-type Yap8p-HA was set to 100 and the ACR3-lacZ induction levels in the presence of the Yap8p mutants are given relative to that of wild-type Yap8p-HA. Error bars represent ± s.d. (D) Yap8p-C132A, C137A and C274A fail to stabilize in the presence of As(III). Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into W303-1A yap8 and Yap8p-HA protein levels were monitored by western blot analysis prior to and 1 hour after addition of 0.2 mM As(III).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Yap8p functions as a homodimer in vivo. (A) Phenotypes of wild-type W303-1A cells expressing Yap8p-HA cysteine to alanine mutants. Plasmids containing wild-type or mutant forms of Yap8p-HA were transformed into wild-type cells and growth of the transformants was scored as above. (B) -galactosidase assays. The W303-1A wild type was co-transformed with a plasmid containing the indicated version of Yap8p-HA and a plasmid containing the ACR3-promoter-lacZ fusion gene. Transformants were assayed for -galactosidase activity as described in Materials and Methods. ACR3-lacZ induction levels were calculated by comparing -galactosidase activities in untreated and As(III)-exposed cells (0.1 mM As(III); 4 hours). The induction level of ACR3-lacZ in the wild-type strain with the empty vector was set to 100 and the ACR3-lacZ induction levels in the presence of the indicated Yap8p versions are given relative to that of wild-type cells with the empty vector. Error bars represent ± s.d. (C) Yap8p forms homodimers in vivo. Co-IP assays were performed with cells co-transformed with two epitope-tagged versions of Yap8p (Myc9-Yap8p and Yap8p-HA). Myc9-Yap8p was immunoprecipitated with an anti-Myc antibody, and the presence of wild-type or mutant forms of Yap8p-HA in the precipitates was detected using an anti-HA antibody. Cells were either untreated or exposed to 0.2 mM As(III) for 1 hour as indicated. The upper panel shows expression levels of wild-type and mutant forms of Yap8p-HA in cells coexpressing Myc9-Yap8p (Input) whereas the lower panel shows Co-IP. Proteins were separated by SDS-PAGE and the presence of Yap8p was monitored with anti-Myc and anti-HA antibodies.