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First published online 3 March 2009
doi: 10.1242/jcs.037291

Journal of Cell Science 122, 976-984 (2009)
Published by The Company of Biologists 2009
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The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I

Daniel J. Termine1, Kelley W. Moremen2,3 and Richard N. Sifers1,4,5,*

1 Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
4 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
5 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
3 Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA


Figure 1
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  		Fig. 1. The mammalian UPR enhances glycoprotein folding and ERAD. Newly synthesized glycoproteins undergo folding attempts in the ER. The re-addition of glucose (+Glc) to N-linked glycans can help promote conformational maturation that leads to deployment (unbroken horizontal green arrows). Under basal conditions, prolonged ER residence can lead to the removal of {alpha}1,2-linked mannose units (–Man) by ERManI, which, together with nonnative protein structure, can initiate entrance into ERAD (unbroken vertical red arrows). A subsequent series of requisite events eventually leads to the addition of ubiquitin (+Ubq) to the polypeptide, which promotes dislocation into the cytosol and degradation by 26S proteasomes. The accumulation of unfolded proteins can evoke the UPR, which transmits signals between the ER and nucleus (curved arrows) to alleviate the situation. The Ire-Xbp1 branch initially enhances protein folding through the transcriptional induction of ER chaperones (broken green arrow), and then boosts glycoprotein ERAD through a process that coincides with the transcriptional elevation of EDEMs.

 

Figure 2
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  		Fig. 2. Post-translational stabilization of transfected human ERManI during the mammalian UPR. (A) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected human ERManI immunoprecipitated from HEK293 cells following 90 minutes of chase. Cells were either not transfected (Co) or were transfected with DNA constructs that encode either {alpha}1-antitrypsin variant null (Hong Kong) (Nhk) or a spliced variant of Xbp1 [Xbp1(s)]. (B) Immunoblots for transfected {alpha}1-antitrypsin variant null (Hong Kong) (Nhk), Xbp1(s) and endogenous BiP from Nonidet P-40 cell extracts under steady-state conditions (top panel) from HEK293 cells transfected with either NHK of Xbp1(s). Immunoblotting for endogenous β-actin served as loading control (bottom panel). (C) Immunoblot of transfected human ERManI from Nonidet P-40 cell extracts under steady-state conditions (top panel). HEK293 cells were transfected with ERManI, and endogenous EDEM1 co-transfected with DNA constructs that encode either {alpha}1-antitrypsin variant null (Hong Kong) (Nhk), or a spliced variant of Xbp1 [Xbp1(s)]. Immunoblotting for endogenous β-actin served as loading control (bottom panel).

 

Figure 3
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  		Fig. 3. Endogenous EDEM1 contributes to the UPR-mediated stabilization of ERManI. (A) Commercially available siRNA (Ambion) were tested for their ability to knock down the expression of endogenous human EDEM1 in HEK293 cells, as demonstrated by immunoblotting of Nonidet P-40 cell lysates. The siRNA identity and concentration are designated (top panel). (B) HEK293 cells were transfected with (+) either human ERManI alone, or in combination with Xbp1(s) and EDEM1 siRNA #64i, as depicted. Immunoblot of transfected Xbp1(s), transfected human ERManI and endogenous EDEM1 from Nonidet P-40 cell extracts under steady-state conditions. Immunoblotting for endogenous β-actin served as loading control.

 

Figure 4
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  		Fig. 4. Co-transfection of EDEM1-HA suppresses proteolytic downregulation of ERManI. (A) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected human ERManI in HEK293 cells without additions (Co) or with (+) co-transfected EDEM1-HA. ERManI was immunoprecipitated from NP-40 cell lysates immediately after the pulse and after 20, 40, 60 and 90 minutes of chase. (B) Quantification of transfected recombinant human ERManI in control (Co) cells, and in those co-transfected with EDEM1 (+EDEM1). (C) HEK293 cells transfected with empty vector (EV) or co-transfected with ERManI and EDEM1-HA (from above). ERManI and EDEM1-HA were immunoprecipitated from equal aliquots of the Nonidet-P40 cell lysate and detected by immunoblotting with the opposite antisera (top panels). Immunoblotting of crude cell lysates detected proteins in the input. The immunoblot of endogenous β-actin demonstrates equal loading of cell lysates (bottom panels).

 

Figure 5
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  		Fig. 5. Endogenous ERManI, but not EDEM1, contributes to the basal GERAD of {alpha}1-antitrypsin PI Z. (A) Commercially available siRNA (Ambion) were tested for their ability to knock down the expression of transfected recombinant human ERManI, as demonstrated by immunoblotting of Nonidet P-40 cell lysates. The siRNA identity and concentration are designated (top panel). Immunoblotting for endogenous β-actin served as loading control (bottom panel). (B) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected {alpha}1-antitrypsin variant PI Z following immunoprecipitation from HEK293 cells (Co) (top panel). As indicated, cells were co-transfected with either #52i to knock down endogenous ERManI (middle panel) or #64i (from Fig. 3B) to knock down endogenous EDEM1 (bottom panel). The horizontal arrows indicate the migration of variant PI Z before and after the trimming of asparagine-linked oligosaccharides. (C) Quantification of intracellular PI Z in control (Co) cells, and in those treated with #52i or #64i at appropriate concentrations. The error bars represent one standard deviation, and represent data generated from at least five experiments.

 

Figure 6
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  		Fig. 6. Transfected EDEM1-HA requires endogenous ERManI to accelerate GERAD of variant PI Z. (A) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected {alpha}1-antitrypsin variant PI Z following immunoprecipitation from HEK293 cells (Co) (top panel). As indicated, cells were co-transfected with ERManI (middle panel) or with ERManI plus #64i to knock down endogenous EDEM1 (bottom panel). An immunoblot of transfected ERManI is shown. (B) Quantification of intracellular radiolabeled PI Z in control (Co) cells, and in those co-transfected with recombinant human ERManI (+ERManI) or co-transfected with ERManI and treated with #64i. (C) HEK293 cells were transfected with variant PI Z alone (Co), or co-transfected with EDEM1-HA (middle panel) or with EDEM1-HA plus #52i to knock down endogenous ERManI (bottom panel). The horizontal arrows indicate the migration of variant PI Z before and after the trimming of asparagine-linked oligosaccharides. An immunoblot of transfected EDEM1-HA is shown. (D) Quantification of intracellular PI Z in control (Co) cells, and in those transfected with EDEM1-HA, or with EDEM1-HA and treated with #52i. The error bars represent one standard deviation, and represent data generated from at least five experiments.

 

Figure 7
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  		Fig. 7. EDEM1 does not require intrinsic mannosidase activity to support ERManI-mediated acceleration of GERAD. (A) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected {alpha}1-antitrypsin variant PI Z following immunoprecipitation from HEK293 cells (Co) (top panel), or co-transfected with either EDEM1(E220A)-HA (middle panel) or EDEM1-HA (bottom panel). The horizontal arrows indicate the migration of radiolabeled PI Z before and after the trimming of asparagine-linked oligosaccharides. Immunoblots for transfected EDEM1-HA (E220A) and EDEM1-HA are shown. A graph demonstrates the quantification of intracellular PI Z remaining in control (Co) cells, and in those transfected with either EDEM1(E220A) or EDEM1-HA. (B) Metabolic pulse-chase radiolabeling and fluorographic detection of transfected human ERManI in HEK293 cells without additions (Co) (top panel), or co-transfected with EDEM1(E220A)-HA (middle panel) or EDEM1-HA (bottom panel). A graph demonstrates the quantification of the intracellular transfected human ERManI remaining in control (Co) cells, and in those transfected with either EDEM1(E220A) or EDEM1-HA. The error bars represent one standard deviation, and represent data generated from at least five experiments. (C) HEK293 cells either not transfected (lane 1), or co-transfected with ERManI and EDEM1(E220A)-HA (lane 2), or with ERManI and EDEM1-HA (lane 3) (from B). EDEM1 was immunoprecipitated from Nonidet-P40 cell lysates and detected by immunoblotting with antiserum against the HA tag (top panel). Immunoblotting of crude cell lysates shows proteins in the input. The immunoblot of endogenous β-actin demonstrates equal loading (bottom panels).

 

Figure 8
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  		Fig. 8. Proposed anatomy of the Ire1-Xbp1 signaling circuitry as it relates to the enhancement of GERAD. The accumulation of unfolded proteins, which leads to ER stress, evokes the UPR, which transmits signals between the ER and nucleus to alleviate the situation. Key elements that contribute to the Ire1-Xbp1 signaling circuitry as it relates to the enhancement of GERAD include initiation, propagation and completion. Key components that contribute to the alleviation of ER stress, via the boosting of GERAD, are depicted.

 

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© The Company of Biologists Ltd 2009