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E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

Ferreira RT, Menezes RA, Rodrigues-Pousada C - Biol Open (2015)

Bottom Line: Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels.Thus, our data disclose a novel Ufd2 role beyond degradation.This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, Oeiras 2781-901, Portugal.

No MeSH data available.


Related in: MedlinePlus

Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as ***P<0.001. (C) Ufd2 protein levels increase during arsenic stress. BY4742 cells expressing Ufd2-HA were treated with 2 mM As(V) or 1.5 mM As(III) and harvested at the indicated time-points. SD, control. Immunoblottings were performed using anti-HA and anti-Pgk1 antibodies. Pgk1 was used as loading control. The graphs represent relative Ufd2 levels (AU, Arbitrary Units). Representative experiments are shown.
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BIO010405F3: Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as ***P<0.001. (C) Ufd2 protein levels increase during arsenic stress. BY4742 cells expressing Ufd2-HA were treated with 2 mM As(V) or 1.5 mM As(III) and harvested at the indicated time-points. SD, control. Immunoblottings were performed using anti-HA and anti-Pgk1 antibodies. Pgk1 was used as loading control. The graphs represent relative Ufd2 levels (AU, Arbitrary Units). Representative experiments are shown.

Mentions: The results above suggest that Ufd2 may play a role in cells exposed to arsenic stress. To characterize the mechanisms by which Ufd2 is implicated in arsenic stress responses, we first evaluated its requirement to arsenic tolerance. We have therefore carried out phenotypic growth assays as well as growth curves of the WT and isogenic ufd2 mutant strains, either in the absence or presence of both As(V) or As(III). UFD2 gene is not essential for yeast cell viability (Bohm et al., 2011), however its deletion leads to a slight growth impairment in the control condition (Fig. 3A), as previously reported (Yoshikawa et al., 2011; Marek and Korona, 2013). Remarkably, ufd2 displayed sensitivity to both As(V) and As(III) stresses (Fig. 3A), which is restored after the reintroduction of an episomal copy of UFD2 in the mutant strain (supplementary material Fig. S2). In agreement with the notion that As(III) is more toxic than As(V) (Ratnaike, 2003), we also noted that UFD2 deletion severely impairs cell growth in the presence of As(III). These results bring to light a novel role for Ufd2 in yeast arsenic adaptation.Fig. 3.


E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

Ferreira RT, Menezes RA, Rodrigues-Pousada C - Biol Open (2015)

Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as ***P<0.001. (C) Ufd2 protein levels increase during arsenic stress. BY4742 cells expressing Ufd2-HA were treated with 2 mM As(V) or 1.5 mM As(III) and harvested at the indicated time-points. SD, control. Immunoblottings were performed using anti-HA and anti-Pgk1 antibodies. Pgk1 was used as loading control. The graphs represent relative Ufd2 levels (AU, Arbitrary Units). Representative experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582114&req=5

BIO010405F3: Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as ***P<0.001. (C) Ufd2 protein levels increase during arsenic stress. BY4742 cells expressing Ufd2-HA were treated with 2 mM As(V) or 1.5 mM As(III) and harvested at the indicated time-points. SD, control. Immunoblottings were performed using anti-HA and anti-Pgk1 antibodies. Pgk1 was used as loading control. The graphs represent relative Ufd2 levels (AU, Arbitrary Units). Representative experiments are shown.
Mentions: The results above suggest that Ufd2 may play a role in cells exposed to arsenic stress. To characterize the mechanisms by which Ufd2 is implicated in arsenic stress responses, we first evaluated its requirement to arsenic tolerance. We have therefore carried out phenotypic growth assays as well as growth curves of the WT and isogenic ufd2 mutant strains, either in the absence or presence of both As(V) or As(III). UFD2 gene is not essential for yeast cell viability (Bohm et al., 2011), however its deletion leads to a slight growth impairment in the control condition (Fig. 3A), as previously reported (Yoshikawa et al., 2011; Marek and Korona, 2013). Remarkably, ufd2 displayed sensitivity to both As(V) and As(III) stresses (Fig. 3A), which is restored after the reintroduction of an episomal copy of UFD2 in the mutant strain (supplementary material Fig. S2). In agreement with the notion that As(III) is more toxic than As(V) (Ratnaike, 2003), we also noted that UFD2 deletion severely impairs cell growth in the presence of As(III). These results bring to light a novel role for Ufd2 in yeast arsenic adaptation.Fig. 3.

Bottom Line: Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels.Thus, our data disclose a novel Ufd2 role beyond degradation.This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, Oeiras 2781-901, Portugal.

No MeSH data available.


Related in: MedlinePlus