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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 Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant 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). (B) Yap8 is destabilized in the ufd2 mutant. 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 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2U-boxΔ mutant strains carrying the GAL1promoterMPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti-c-myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2. Exponential phase BY4742 WT, yap8, ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D 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 or ***P<0.001. (F) YAP8 overexpression recovers ufd2 growth in cells exposed to arsenic stress. Exponential phase BY4742 WT and the ufd2 mutant strain overexpressing YAP8 or the vector alone were serially diluted and spotted onto SD media supplemented with 1.5 As(III) or 2 mM As(V). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
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BIO010405F4: Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant 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). (B) Yap8 is destabilized in the ufd2 mutant. 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 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2U-boxΔ mutant strains carrying the GAL1promoterMPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti-c-myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2. Exponential phase BY4742 WT, yap8, ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D 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 or ***P<0.001. (F) YAP8 overexpression recovers ufd2 growth in cells exposed to arsenic stress. Exponential phase BY4742 WT and the ufd2 mutant strain overexpressing YAP8 or the vector alone were serially diluted and spotted onto SD media supplemented with 1.5 As(III) or 2 mM As(V). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.

Mentions: Ufd2 was shown to be involved in proteasomal degradation pathways (Liu et al., 2010). On the other hand, our data suggest that interaction of Ufd2 with Yap8 protects yeast cells against the toxicity of As compounds. To clarify the functional relationship between Ufd2 and Yap8, we have monitored the kinetics of Yap8 fused to HA epitope (Yap8-HA) induced by As(III) in WT and ufd2 strains. As shown in Fig. 4A, ufd2 displayed lower Yap8 levels when compared to WT cells indicating that Yap8 stability may be compromised in the absence of Ufd2.Fig. 4.


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 Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant 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). (B) Yap8 is destabilized in the ufd2 mutant. 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 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2U-boxΔ mutant strains carrying the GAL1promoterMPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti-c-myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2. Exponential phase BY4742 WT, yap8, ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D 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 or ***P<0.001. (F) YAP8 overexpression recovers ufd2 growth in cells exposed to arsenic stress. Exponential phase BY4742 WT and the ufd2 mutant strain overexpressing YAP8 or the vector alone were serially diluted and spotted onto SD media supplemented with 1.5 As(III) or 2 mM As(V). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
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BIO010405F4: Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant 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). (B) Yap8 is destabilized in the ufd2 mutant. 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 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2U-boxΔ mutant strains carrying the GAL1promoterMPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti-c-myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2. Exponential phase BY4742 WT, yap8, ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D 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 or ***P<0.001. (F) YAP8 overexpression recovers ufd2 growth in cells exposed to arsenic stress. Exponential phase BY4742 WT and the ufd2 mutant strain overexpressing YAP8 or the vector alone were serially diluted and spotted onto SD media supplemented with 1.5 As(III) or 2 mM As(V). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown.
Mentions: Ufd2 was shown to be involved in proteasomal degradation pathways (Liu et al., 2010). On the other hand, our data suggest that interaction of Ufd2 with Yap8 protects yeast cells against the toxicity of As compounds. To clarify the functional relationship between Ufd2 and Yap8, we have monitored the kinetics of Yap8 fused to HA epitope (Yap8-HA) induced by As(III) in WT and ufd2 strains. As shown in Fig. 4A, ufd2 displayed lower Yap8 levels when compared to WT cells indicating that Yap8 stability may be compromised in the absence of Ufd2.Fig. 4.

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