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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

Arsenic triggers Yap8 stabilization circumventing proteasomal degradation. (A) Wild type cells exposed to arsenite exhibit enhanced Yap8 levels. BY4742 wild type (WT) cells expressing Yap8-HA were left untreated (SD) or exposed to 1.5 mM As(III) alone or in combination with 100 µM MG132 and DMSO, for 90 min. The graph represents relative Yap8 levels normalized against Pgk1 levels (arbitrary units, AU). (B) As(III) triggers Yap8 stabilization. Cells were first exposed to 1.5 mM As(III) for 30 min, cycloheximide (CHX) was then added to a final concentration of 0.1 mg/ml up to 240 min and protein extracts were subjected to immunoblotting using a anti-HA antibody. Pgk1 was used as loading control. The graph represents the percentage of remaining Yap8 after CHX addition. Left panel: As(III) was maintained in the medium; estimated Yap8 half-life >240 min. Right panel: As(III) was removed from the medium before CHX addition; estimated Yap8 half-life is 94 min. The arrow indicates the removal of As(III). Representative experiments are shown.
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BIO010405F1: Arsenic triggers Yap8 stabilization circumventing proteasomal degradation. (A) Wild type cells exposed to arsenite exhibit enhanced Yap8 levels. BY4742 wild type (WT) cells expressing Yap8-HA were left untreated (SD) or exposed to 1.5 mM As(III) alone or in combination with 100 µM MG132 and DMSO, for 90 min. The graph represents relative Yap8 levels normalized against Pgk1 levels (arbitrary units, AU). (B) As(III) triggers Yap8 stabilization. Cells were first exposed to 1.5 mM As(III) for 30 min, cycloheximide (CHX) was then added to a final concentration of 0.1 mg/ml up to 240 min and protein extracts were subjected to immunoblotting using a anti-HA antibody. Pgk1 was used as loading control. The graph represents the percentage of remaining Yap8 after CHX addition. Left panel: As(III) was maintained in the medium; estimated Yap8 half-life >240 min. Right panel: As(III) was removed from the medium before CHX addition; estimated Yap8 half-life is 94 min. The arrow indicates the removal of As(III). Representative experiments are shown.

Mentions: Arsenic stress responses in S. cerevisiae require the AP-1 like transcription factor Yap8, which drives the expression of genes involved in As detoxification processes (Menezes et al., 2004). Aiming at elucidating further the mechanisms underlying Yap8 regulation, we analysed by immunoblotting the levels and the stability of HA-tagged Yap8 in wild type (WT) cells, either in the presence or the absence of arsenite (Fig. 1). Yap8-HA levels were shown to be low in cells incubated under control conditions, and increased in response to 90 min treatment with As(III) (Fig. 1A, lanes 1 and 2). Furthermore, inhibition of proteasome activity with MG132 led to an increase of Yap8-HA levels under non-inducing conditions (Fig. 1A, lanes 1 and 3), but not in the presence of As(III), as indicated by cell co-treatment with MG132 and As(III) (Fig. 1A, lanes 2 and 4). These data indicate that Yap8 is not degraded by proteasome under arsenic stress conditions. In DMSO-treated cells, Yap8 levels are comparable to those observed in the control condition (Fig. 1A, lanes 1 and 5). We have then determined Yap8 stability in the presence and absence of As by using the protein synthesis inhibitor cycloheximide (CHX). For that, cells were first pre-treated with 1.5 mM arsenite for 30 min followed by CHX treatment up to 240 min. It is clear that Yap8 levels were enhanced during this period (Fig. 1B, left panel) in contrast to what does occur if As was completely removed from the medium before CHX treatment (Fig. 1B, right panel). Measurement of Yap8 half-life in both conditions revealed that it is strongly reduced in the absence of As (>240 min vs 94 min).Fig. 1.


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)

Arsenic triggers Yap8 stabilization circumventing proteasomal degradation. (A) Wild type cells exposed to arsenite exhibit enhanced Yap8 levels. BY4742 wild type (WT) cells expressing Yap8-HA were left untreated (SD) or exposed to 1.5 mM As(III) alone or in combination with 100 µM MG132 and DMSO, for 90 min. The graph represents relative Yap8 levels normalized against Pgk1 levels (arbitrary units, AU). (B) As(III) triggers Yap8 stabilization. Cells were first exposed to 1.5 mM As(III) for 30 min, cycloheximide (CHX) was then added to a final concentration of 0.1 mg/ml up to 240 min and protein extracts were subjected to immunoblotting using a anti-HA antibody. Pgk1 was used as loading control. The graph represents the percentage of remaining Yap8 after CHX addition. Left panel: As(III) was maintained in the medium; estimated Yap8 half-life >240 min. Right panel: As(III) was removed from the medium before CHX addition; estimated Yap8 half-life is 94 min. The arrow indicates the removal of As(III). Representative experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4582114&req=5

BIO010405F1: Arsenic triggers Yap8 stabilization circumventing proteasomal degradation. (A) Wild type cells exposed to arsenite exhibit enhanced Yap8 levels. BY4742 wild type (WT) cells expressing Yap8-HA were left untreated (SD) or exposed to 1.5 mM As(III) alone or in combination with 100 µM MG132 and DMSO, for 90 min. The graph represents relative Yap8 levels normalized against Pgk1 levels (arbitrary units, AU). (B) As(III) triggers Yap8 stabilization. Cells were first exposed to 1.5 mM As(III) for 30 min, cycloheximide (CHX) was then added to a final concentration of 0.1 mg/ml up to 240 min and protein extracts were subjected to immunoblotting using a anti-HA antibody. Pgk1 was used as loading control. The graph represents the percentage of remaining Yap8 after CHX addition. Left panel: As(III) was maintained in the medium; estimated Yap8 half-life >240 min. Right panel: As(III) was removed from the medium before CHX addition; estimated Yap8 half-life is 94 min. The arrow indicates the removal of As(III). Representative experiments are shown.
Mentions: Arsenic stress responses in S. cerevisiae require the AP-1 like transcription factor Yap8, which drives the expression of genes involved in As detoxification processes (Menezes et al., 2004). Aiming at elucidating further the mechanisms underlying Yap8 regulation, we analysed by immunoblotting the levels and the stability of HA-tagged Yap8 in wild type (WT) cells, either in the presence or the absence of arsenite (Fig. 1). Yap8-HA levels were shown to be low in cells incubated under control conditions, and increased in response to 90 min treatment with As(III) (Fig. 1A, lanes 1 and 2). Furthermore, inhibition of proteasome activity with MG132 led to an increase of Yap8-HA levels under non-inducing conditions (Fig. 1A, lanes 1 and 3), but not in the presence of As(III), as indicated by cell co-treatment with MG132 and As(III) (Fig. 1A, lanes 2 and 4). These data indicate that Yap8 is not degraded by proteasome under arsenic stress conditions. In DMSO-treated cells, Yap8 levels are comparable to those observed in the control condition (Fig. 1A, lanes 1 and 5). We have then determined Yap8 stability in the presence and absence of As by using the protein synthesis inhibitor cycloheximide (CHX). For that, cells were first pre-treated with 1.5 mM arsenite for 30 min followed by CHX treatment up to 240 min. It is clear that Yap8 levels were enhanced during this period (Fig. 1B, left panel) in contrast to what does occur if As was completely removed from the medium before CHX treatment (Fig. 1B, right panel). Measurement of Yap8 half-life in both conditions revealed that it is strongly reduced in the absence of As (>240 min vs 94 min).Fig. 1.

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