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gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum.

Zhang T, Xu Y, Liu Y, Ye Y - Mol. Biol. Cell (2015)

Bottom Line: Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD).Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex.We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.

No MeSH data available.


Related in: MedlinePlus

Hrd1 but not gp78 is required for ubiquitination of ERAD substrates. (A and B) The model ERAD substrates MHC 1-147-FLAG (A) and TCRα-YFP-FLAG (B) were cotransfected with a construct expressing HA-tagged ubiquitin in either control or hrd1 CRIPSR cell. The cells were treated with either DMSO as a control or with the proteasome inhibitor MG132 (10 μM, 15 h). Substrates immunoprecipitated from the cell extracts under denaturing conditions were analyzed by immunoblotting. The anti-HA blot reveals ubiquitinated substrate and the anti-FLAG blot shows the nonubiquitinated glycosylated and deglycosylated substrates. (C) As in A, except that cells treated with the indicated shRNA constructs were used. (D) As in B, except that cells treated with the indicated shRNA constructs were used. (E) Endoglycosidase H treatment of the indicated cell extract reveals the glycosylation pattern of MHC 1-147 in the indicated CRISPR cells that have been exposed to a proteasome inhibitor.
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Figure 4: Hrd1 but not gp78 is required for ubiquitination of ERAD substrates. (A and B) The model ERAD substrates MHC 1-147-FLAG (A) and TCRα-YFP-FLAG (B) were cotransfected with a construct expressing HA-tagged ubiquitin in either control or hrd1 CRIPSR cell. The cells were treated with either DMSO as a control or with the proteasome inhibitor MG132 (10 μM, 15 h). Substrates immunoprecipitated from the cell extracts under denaturing conditions were analyzed by immunoblotting. The anti-HA blot reveals ubiquitinated substrate and the anti-FLAG blot shows the nonubiquitinated glycosylated and deglycosylated substrates. (C) As in A, except that cells treated with the indicated shRNA constructs were used. (D) As in B, except that cells treated with the indicated shRNA constructs were used. (E) Endoglycosidase H treatment of the indicated cell extract reveals the glycosylation pattern of MHC 1-147 in the indicated CRISPR cells that have been exposed to a proteasome inhibitor.

Mentions: Next we tested whether both gp78 and Hrd1 were required for substrate polyubiquitination. We first expressed MHC 1-147 in control and Hrd1 CRISPR cells together with hemagglutinin (HA)-tagged ubiquitin. We then treated these cells with DMSO as a control or the proteasome inhibitor MG132 and immunoprecipitated MHC 1-147 from cell extracts under denaturing conditions. Immunoblotting with FLAG antibodies showed that inhibition of the proteasome caused accumulation of MHC 1-147 in both glycosylated and deglycosylated forms (Figure 4A, bottom panel, lane 3 vs. lane 1). The deglycosylated MHC 1-147 must have arrived in the cytosol, because the N-glycanase that processes the sugar chain is cytosolic (Blom et al., 2004). The glycosylated form accumulated mostly in the ER due to a retrotranslocation backup, although a fraction might have reached the cytosolic side of the ER membrane, but the glycan had not been removed. Knockout of hrd1 caused MHC 1-147 to accumulate only in the glycosylated form (Figure 4A, lane 2). When control and hrd1 CRISPR cells were treated with MG132, glycosylated MHC 1-147 was accumulated similarly, but the amount of deglycosylated MHC 1-147 was significantly less in hrd1 knockout cells than in control cells (Figure 4A, lane 4 vs. lane 3). These observations are consistent with the proposed function of Hrd1 in retrotranslocation. Immunoblotting with HA antibodies detected ubiquitinated MHC 1-147 in control cells, and as expected, ubiquitinated MHC 1-147 was increased when degradation was blocked by the proteasome inhibitor MG132 (Figure 4A, lane 3 vs. lane 1). However, the level of ubiquitinated MHC 1-147 was significantly reduced in Hrd1 CRISPR cells compared with control cells under both untreated and MG132-treated conditions (Figure 4A, lane 2 vs. lane 1 and lane 4 vs. lane 3). Similar results were obtained with the membrane ERAD substrate TCRα (Figure 4B). We next examined the role of gp78 in these ERAD processes by transiently knocking down the gp78 expression. Surprisingly, knockdown of gp78 neither reduced the level of deglycosylated MHC 1-147 nor inhibited MHC 1-147 ubiquitination (Figure 4, C, lane 4 vs. lane 3, and D). Likewise, ubiquitination of TCRα was also not inhibited by gp78 knockdown (Figure 4E). Thus, for substrates whose degradation requires both Hrd1 and gp78, only Hrd1 but not gp78 is essential for ubiquitination.


gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum.

Zhang T, Xu Y, Liu Y, Ye Y - Mol. Biol. Cell (2015)

Hrd1 but not gp78 is required for ubiquitination of ERAD substrates. (A and B) The model ERAD substrates MHC 1-147-FLAG (A) and TCRα-YFP-FLAG (B) were cotransfected with a construct expressing HA-tagged ubiquitin in either control or hrd1 CRIPSR cell. The cells were treated with either DMSO as a control or with the proteasome inhibitor MG132 (10 μM, 15 h). Substrates immunoprecipitated from the cell extracts under denaturing conditions were analyzed by immunoblotting. The anti-HA blot reveals ubiquitinated substrate and the anti-FLAG blot shows the nonubiquitinated glycosylated and deglycosylated substrates. (C) As in A, except that cells treated with the indicated shRNA constructs were used. (D) As in B, except that cells treated with the indicated shRNA constructs were used. (E) Endoglycosidase H treatment of the indicated cell extract reveals the glycosylation pattern of MHC 1-147 in the indicated CRISPR cells that have been exposed to a proteasome inhibitor.
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Related In: Results  -  Collection

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Figure 4: Hrd1 but not gp78 is required for ubiquitination of ERAD substrates. (A and B) The model ERAD substrates MHC 1-147-FLAG (A) and TCRα-YFP-FLAG (B) were cotransfected with a construct expressing HA-tagged ubiquitin in either control or hrd1 CRIPSR cell. The cells were treated with either DMSO as a control or with the proteasome inhibitor MG132 (10 μM, 15 h). Substrates immunoprecipitated from the cell extracts under denaturing conditions were analyzed by immunoblotting. The anti-HA blot reveals ubiquitinated substrate and the anti-FLAG blot shows the nonubiquitinated glycosylated and deglycosylated substrates. (C) As in A, except that cells treated with the indicated shRNA constructs were used. (D) As in B, except that cells treated with the indicated shRNA constructs were used. (E) Endoglycosidase H treatment of the indicated cell extract reveals the glycosylation pattern of MHC 1-147 in the indicated CRISPR cells that have been exposed to a proteasome inhibitor.
Mentions: Next we tested whether both gp78 and Hrd1 were required for substrate polyubiquitination. We first expressed MHC 1-147 in control and Hrd1 CRISPR cells together with hemagglutinin (HA)-tagged ubiquitin. We then treated these cells with DMSO as a control or the proteasome inhibitor MG132 and immunoprecipitated MHC 1-147 from cell extracts under denaturing conditions. Immunoblotting with FLAG antibodies showed that inhibition of the proteasome caused accumulation of MHC 1-147 in both glycosylated and deglycosylated forms (Figure 4A, bottom panel, lane 3 vs. lane 1). The deglycosylated MHC 1-147 must have arrived in the cytosol, because the N-glycanase that processes the sugar chain is cytosolic (Blom et al., 2004). The glycosylated form accumulated mostly in the ER due to a retrotranslocation backup, although a fraction might have reached the cytosolic side of the ER membrane, but the glycan had not been removed. Knockout of hrd1 caused MHC 1-147 to accumulate only in the glycosylated form (Figure 4A, lane 2). When control and hrd1 CRISPR cells were treated with MG132, glycosylated MHC 1-147 was accumulated similarly, but the amount of deglycosylated MHC 1-147 was significantly less in hrd1 knockout cells than in control cells (Figure 4A, lane 4 vs. lane 3). These observations are consistent with the proposed function of Hrd1 in retrotranslocation. Immunoblotting with HA antibodies detected ubiquitinated MHC 1-147 in control cells, and as expected, ubiquitinated MHC 1-147 was increased when degradation was blocked by the proteasome inhibitor MG132 (Figure 4A, lane 3 vs. lane 1). However, the level of ubiquitinated MHC 1-147 was significantly reduced in Hrd1 CRISPR cells compared with control cells under both untreated and MG132-treated conditions (Figure 4A, lane 2 vs. lane 1 and lane 4 vs. lane 3). Similar results were obtained with the membrane ERAD substrate TCRα (Figure 4B). We next examined the role of gp78 in these ERAD processes by transiently knocking down the gp78 expression. Surprisingly, knockdown of gp78 neither reduced the level of deglycosylated MHC 1-147 nor inhibited MHC 1-147 ubiquitination (Figure 4, C, lane 4 vs. lane 3, and D). Likewise, ubiquitination of TCRα was also not inhibited by gp78 knockdown (Figure 4E). Thus, for substrates whose degradation requires both Hrd1 and gp78, only Hrd1 but not gp78 is essential for ubiquitination.

Bottom Line: Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD).Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex.We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.

No MeSH data available.


Related in: MedlinePlus