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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

Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA 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 using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4, rad23 and dsk2 mutant cells. 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. No significant statistical differences were observed.
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BIO010405F6: Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA 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 using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4, rad23 and dsk2 mutant cells. 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. No significant statistical differences were observed.

Mentions: As to determine whether Ufd2 role on Yap8 stabilization is connected to UPP we next investigated the requirement of the UPP components Ubc4, Rad23 and Dsk2 to Yap8 stability under arsenic stress conditions. The E2 enzyme Ubc4 is upstream of Ufd2 in the ubiquitination process of proteins targeted to proteasome (Jentsch et al., 1990; Sommer and Seufert, 1992) whereas Rad23 and Dsk2 are Ufd2-downstream players bridging ubiquitinated proteins to the proteasome (Chen and Madura, 2002; Funakoshi et al., 2002; Kim et al., 2004). Importantly, the concerted action of Ufd2, Rad23 and Dsk2 in the degradation of Mps1 kinase was already described (Liu et al., 2011). In contrast to the reduced Yap8 stability observed in the ufd2 strain, Yap8 half-life was found to be higher than the WT strain in the ubc4, rad23 and dsk2 mutants challenged with arsenite (Fig. 6A-C). Furthermore, arsenite-mediated upregulation of ACR3 is not significantly affected in these mutants (Fig. 6D) indicating that Ubc4, Rad23 and Dsk2 are dispensable for Ufd2-mediated Yap8 stabilization and transcriptional activity. These results are also consistent with the observation that Yap8 is stabilized in mutants with defective proteasomal activity (Di and Tamas, 2007).Fig. 6.


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)

Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA 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 using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4, rad23 and dsk2 mutant cells. 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. No significant statistical differences were observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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BIO010405F6: Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA 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 using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4, rad23 and dsk2 mutant cells. 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. No significant statistical differences were observed.
Mentions: As to determine whether Ufd2 role on Yap8 stabilization is connected to UPP we next investigated the requirement of the UPP components Ubc4, Rad23 and Dsk2 to Yap8 stability under arsenic stress conditions. The E2 enzyme Ubc4 is upstream of Ufd2 in the ubiquitination process of proteins targeted to proteasome (Jentsch et al., 1990; Sommer and Seufert, 1992) whereas Rad23 and Dsk2 are Ufd2-downstream players bridging ubiquitinated proteins to the proteasome (Chen and Madura, 2002; Funakoshi et al., 2002; Kim et al., 2004). Importantly, the concerted action of Ufd2, Rad23 and Dsk2 in the degradation of Mps1 kinase was already described (Liu et al., 2011). In contrast to the reduced Yap8 stability observed in the ufd2 strain, Yap8 half-life was found to be higher than the WT strain in the ubc4, rad23 and dsk2 mutants challenged with arsenite (Fig. 6A-C). Furthermore, arsenite-mediated upregulation of ACR3 is not significantly affected in these mutants (Fig. 6D) indicating that Ubc4, Rad23 and Dsk2 are dispensable for Ufd2-mediated Yap8 stabilization and transcriptional activity. These results are also consistent with the observation that Yap8 is stabilized in mutants with defective proteasomal activity (Di and Tamas, 2007).Fig. 6.

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