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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 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2U-boxΔ. (C) ACR3 mRNA levels are unaltered in the Ufd2U-boxΔ. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 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<0.05. (D) The Ufd2U-boxΔ mutant is tolerant to arsenic stress. Exponential phase BY4741 WT, ufd2 and Ufd2U-boxΔ cells were serially diluted and spotted onto SC media supplemented or not with 2 mM As(V) or 1.5 mM As(III). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
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BIO010405F5: Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2U-boxΔ. (C) ACR3 mRNA levels are unaltered in the Ufd2U-boxΔ. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 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<0.05. (D) The Ufd2U-boxΔ mutant is tolerant to arsenic stress. Exponential phase BY4741 WT, ufd2 and Ufd2U-boxΔ cells were serially diluted and spotted onto SC media supplemented or not with 2 mM As(V) or 1.5 mM As(III). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.

Mentions: Several reports indicate that Ufd2 U-box motif is essential for its ubiquitination activity (Aravind and Koonin, 2000; Tu et al., 2007). We have therefore investigated whether this activity was also required for Yap8 stabilization and cell tolerance to arsenic stress. BY4741 isogenic strains were used for these experiments since the Ufd2U-boxΔ mutant is available exclusively in this yeast background (Liu et al., 2011). First, it was examined Yap8-HA kinetics in WT, ufd2 and Ufd2U-boxΔ strains induced for 90 min with As(III). Corroborating the data from the BY4742 strain, Yap8 levels were found to be reduced in the ufd2 mutant (Fig. 5A). Remarkably, the Ufd2U-boxΔ mutant displayed similar Yap8 levels as compared to the WT strain indicating that Ufd2 U-box domain is not involved in the regulation of Yap8 protein levels under arsenic stress. Next, Yap8 stability was monitored in the same strains. Cells were induced for 90 min with As(III) after which arsenic was removed and cells were treated with CHX for further 90 min. Supporting previous results obtained in the BY4742 strain (Fig. 4B), Yap8 stability is compromised in the ufd2 mutant (Fig. 5B, half-life 25 min). Notwithstanding, Ufd2U-boxΔ mutant cells displayed similar Yap8 half-life compared to WT cells (67 and 63 min, respectively) confirming that Ufd2 U-box motif is dispensable for Yap8 stabilization during arsenic stress.Fig. 5.


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 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2U-boxΔ. (C) ACR3 mRNA levels are unaltered in the Ufd2U-boxΔ. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 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<0.05. (D) The Ufd2U-boxΔ mutant is tolerant to arsenic stress. Exponential phase BY4741 WT, ufd2 and Ufd2U-boxΔ cells were serially diluted and spotted onto SC media supplemented or not with 2 mM As(V) or 1.5 mM As(III). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
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BIO010405F5: Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2U-boxΔ. (C) ACR3 mRNA levels are unaltered in the Ufd2U-boxΔ. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 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<0.05. (D) The Ufd2U-boxΔ mutant is tolerant to arsenic stress. Exponential phase BY4741 WT, ufd2 and Ufd2U-boxΔ cells were serially diluted and spotted onto SC media supplemented or not with 2 mM As(V) or 1.5 mM As(III). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
Mentions: Several reports indicate that Ufd2 U-box motif is essential for its ubiquitination activity (Aravind and Koonin, 2000; Tu et al., 2007). We have therefore investigated whether this activity was also required for Yap8 stabilization and cell tolerance to arsenic stress. BY4741 isogenic strains were used for these experiments since the Ufd2U-boxΔ mutant is available exclusively in this yeast background (Liu et al., 2011). First, it was examined Yap8-HA kinetics in WT, ufd2 and Ufd2U-boxΔ strains induced for 90 min with As(III). Corroborating the data from the BY4742 strain, Yap8 levels were found to be reduced in the ufd2 mutant (Fig. 5A). Remarkably, the Ufd2U-boxΔ mutant displayed similar Yap8 levels as compared to the WT strain indicating that Ufd2 U-box domain is not involved in the regulation of Yap8 protein levels under arsenic stress. Next, Yap8 stability was monitored in the same strains. Cells were induced for 90 min with As(III) after which arsenic was removed and cells were treated with CHX for further 90 min. Supporting previous results obtained in the BY4742 strain (Fig. 4B), Yap8 stability is compromised in the ufd2 mutant (Fig. 5B, half-life 25 min). Notwithstanding, Ufd2U-boxΔ mutant cells displayed similar Yap8 half-life compared to WT cells (67 and 63 min, respectively) confirming that Ufd2 U-box motif is dispensable for Yap8 stabilization during arsenic stress.Fig. 5.

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