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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

Both gp78 and Hrd1 are required for ERAD of luminal and membrane substrates. (A) Diagram illustrating the model ERAD substrates used in this study. (B) Both gp78 and UbxD8 are required for degradation of MHC 1-147. Cells cotransfected with indicated shRNA constructs and a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting. Graph on the right represents the quantification of the experiment. (C) Guiding sequence used to create hrd1 knockout CRISPR cell. The PAM sequence and the target sequence are colored in red and blue, respectively. Red arrow indicates the predicted Cas9 D10A cutting site. (D) Hrd1 is required for the degradation of MHC 1-147. Cycloheximide chase was performed in control CRISPR and hrd1 knockout CRISPR cells. Where indicated, plasmids expressing WT Hrd1 or a catalytically inactive Hrd1 (C1C3) mutant were cotransfected with MHC 1-147. Whole-cell extracts were analyzed by immunoblotting. (E) Verification of the gp78 CRISPR cells by immunoblotting. (F and G) gp78-deficient CRISPR cells do not have ERAD defects. The steady-state level of MHC 1-147 in either the parental HEK293T cells or the indicated CRISPR clones was analyzed by immunoblotting. Where indicated, cells were treated with the proteasome inhibitor MG132 (10 μm, 15 h). (G) The indicated CRISPR cells transfected with a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting.
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Figure 3: Both gp78 and Hrd1 are required for ERAD of luminal and membrane substrates. (A) Diagram illustrating the model ERAD substrates used in this study. (B) Both gp78 and UbxD8 are required for degradation of MHC 1-147. Cells cotransfected with indicated shRNA constructs and a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting. Graph on the right represents the quantification of the experiment. (C) Guiding sequence used to create hrd1 knockout CRISPR cell. The PAM sequence and the target sequence are colored in red and blue, respectively. Red arrow indicates the predicted Cas9 D10A cutting site. (D) Hrd1 is required for the degradation of MHC 1-147. Cycloheximide chase was performed in control CRISPR and hrd1 knockout CRISPR cells. Where indicated, plasmids expressing WT Hrd1 or a catalytically inactive Hrd1 (C1C3) mutant were cotransfected with MHC 1-147. Whole-cell extracts were analyzed by immunoblotting. (E) Verification of the gp78 CRISPR cells by immunoblotting. (F and G) gp78-deficient CRISPR cells do not have ERAD defects. The steady-state level of MHC 1-147 in either the parental HEK293T cells or the indicated CRISPR clones was analyzed by immunoblotting. Where indicated, cells were treated with the proteasome inhibitor MG132 (10 μm, 15 h). (G) The indicated CRISPR cells transfected with a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting.

Mentions: The physical interaction between the gp78 and the Hrd1 complex (Figure 1) suggested that these enzymes might cooperate with each other in ERAD. Conceptually, the two ligases might act in a linear pathway, or they might function in parallel. To study the functional interplay between gp78 and Hrd1, we sought model ERAD substrates that require both Hrd1 and gp78 for degradation. As a representative of membrane substrates, we chose TCRα (Figure 3A), a type I membrane protein that is mostly unassembled when overexpressed in tissue culture cells. As a result, overexpressed TCRα is rapidly degraded by a mechanism dependent on both gp78 and Hrd1 (Ishikura et al., 2010). Because previously reported luminal ERAD substrates do not require gp78 for efficient degradation, we engineered a truncated major histocompatibility complex (MHC) class I heavy-chain molecule (hereinafter referred to as “MHC 1-147”) that contains only a small luminal segment of the human leukocyte antigen (HLA) A2 allele (Figure 3A). A similar MHC class I mutant was previously established as a luminal ERAD substrate whose degradation requires the Hrd1 complex (Burr et al., 2013). We used short hairpin RNAs (shRNAs) to knock down either gp78 or UbxD8 and tested whether the half-life of MHC 1-147 was affected using a translation shutoff assay. The result suggested that gp78 and UbxD8 are both required for efficient degradation of MHC 1-147 (Figure 3B).


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)

Both gp78 and Hrd1 are required for ERAD of luminal and membrane substrates. (A) Diagram illustrating the model ERAD substrates used in this study. (B) Both gp78 and UbxD8 are required for degradation of MHC 1-147. Cells cotransfected with indicated shRNA constructs and a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting. Graph on the right represents the quantification of the experiment. (C) Guiding sequence used to create hrd1 knockout CRISPR cell. The PAM sequence and the target sequence are colored in red and blue, respectively. Red arrow indicates the predicted Cas9 D10A cutting site. (D) Hrd1 is required for the degradation of MHC 1-147. Cycloheximide chase was performed in control CRISPR and hrd1 knockout CRISPR cells. Where indicated, plasmids expressing WT Hrd1 or a catalytically inactive Hrd1 (C1C3) mutant were cotransfected with MHC 1-147. Whole-cell extracts were analyzed by immunoblotting. (E) Verification of the gp78 CRISPR cells by immunoblotting. (F and G) gp78-deficient CRISPR cells do not have ERAD defects. The steady-state level of MHC 1-147 in either the parental HEK293T cells or the indicated CRISPR clones was analyzed by immunoblotting. Where indicated, cells were treated with the proteasome inhibitor MG132 (10 μm, 15 h). (G) The indicated CRISPR cells transfected with a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting.
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Figure 3: Both gp78 and Hrd1 are required for ERAD of luminal and membrane substrates. (A) Diagram illustrating the model ERAD substrates used in this study. (B) Both gp78 and UbxD8 are required for degradation of MHC 1-147. Cells cotransfected with indicated shRNA constructs and a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting. Graph on the right represents the quantification of the experiment. (C) Guiding sequence used to create hrd1 knockout CRISPR cell. The PAM sequence and the target sequence are colored in red and blue, respectively. Red arrow indicates the predicted Cas9 D10A cutting site. (D) Hrd1 is required for the degradation of MHC 1-147. Cycloheximide chase was performed in control CRISPR and hrd1 knockout CRISPR cells. Where indicated, plasmids expressing WT Hrd1 or a catalytically inactive Hrd1 (C1C3) mutant were cotransfected with MHC 1-147. Whole-cell extracts were analyzed by immunoblotting. (E) Verification of the gp78 CRISPR cells by immunoblotting. (F and G) gp78-deficient CRISPR cells do not have ERAD defects. The steady-state level of MHC 1-147 in either the parental HEK293T cells or the indicated CRISPR clones was analyzed by immunoblotting. Where indicated, cells were treated with the proteasome inhibitor MG132 (10 μm, 15 h). (G) The indicated CRISPR cells transfected with a plasmid expressing MHC 1-147 were treated with cycloheximide for the indicated time points. Cells were directly lysed in the Laemmli buffer, and the whole-cell extracts were analyzed by immunoblotting.
Mentions: The physical interaction between the gp78 and the Hrd1 complex (Figure 1) suggested that these enzymes might cooperate with each other in ERAD. Conceptually, the two ligases might act in a linear pathway, or they might function in parallel. To study the functional interplay between gp78 and Hrd1, we sought model ERAD substrates that require both Hrd1 and gp78 for degradation. As a representative of membrane substrates, we chose TCRα (Figure 3A), a type I membrane protein that is mostly unassembled when overexpressed in tissue culture cells. As a result, overexpressed TCRα is rapidly degraded by a mechanism dependent on both gp78 and Hrd1 (Ishikura et al., 2010). Because previously reported luminal ERAD substrates do not require gp78 for efficient degradation, we engineered a truncated major histocompatibility complex (MHC) class I heavy-chain molecule (hereinafter referred to as “MHC 1-147”) that contains only a small luminal segment of the human leukocyte antigen (HLA) A2 allele (Figure 3A). A similar MHC class I mutant was previously established as a luminal ERAD substrate whose degradation requires the Hrd1 complex (Burr et al., 2013). We used short hairpin RNAs (shRNAs) to knock down either gp78 or UbxD8 and tested whether the half-life of MHC 1-147 was affected using a translation shutoff assay. The result suggested that gp78 and UbxD8 are both required for efficient degradation of MHC 1-147 (Figure 3B).

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