1. Gp78, a Membrane-Anchored Ubiquitin Ligase, Associates with Insig-1 and Couples Sterol-Regulated Ubiquitination to Degradation of HMG CoA Reductase 
Bao-Liang Song, Navdar Sever, Russell A. DeBose-Boyd 
Molecular Cell 
Volume 19, Issue 6, Pages (September 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Ubiquitin-Ligase Activity Associates with Insig-1
(A) Schematic representation of the affinity purification approach used in the current study to identify an Insig-1-associated ubiquitin ligase.
(B) Stock cultures of CHO/pInsig-1-Myc cells were set up on day 0 at 5 × 105 cells per 100 mm dish. On day 2, the cells were switched to medium A containing 5% LPDS, 10 μM sodium compactin, and 50 μM mevalonate. After incubation for 16 hr at 37°C, cells were refed identical medium supplemented with 10 μg/ml lanosterol and 10 μM MG-132 and incubated for an addition 2 hr at 37°C. Cells were then harvested and permeabilized, and detergent lysates were prepared. Immunoprecipitation was carried out with either anti-Myc (lanes 1–4)- or anti-T7 (lanes 5–8)-coupled agarose beads as described in Experimental Procedures. The immunoprecipitates were then subjected to incubation in buffer containing an ATP-regenerating system, 0.1 mg/ml FLAG-ubiquitin, and 2.5 μg purified E1 as indicated. Reactions were carried out for 30 min at 37°C, after which they were terminated by centrifugation. The resulting pellets were subjected to SDS-PAGE, and immunoblot analysis was carried out with 1 μg/ml IgG-M2 (against FLAG-ubiquitin) or IgG-9E10 (against Insig-1). Filters were exposed to film at room temperature for 15 s.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 The Membrane Domain of gp78 Mediates its Binding to Insig-1
(A) Schematic representation of the domain structure of gp78.
(B–E) Stock cultures of CHO-K1 cells were set up for experiments at 5 × 105 cells per 60 mm dish. On day 1, cells were transfected with the indicated expression plasmids in 2 ml of medium A containing 5% LPDS. The total amount of DNA in each lane was adjusted to 3 μg/dish by the addition of pcDNA3 mock vector. Six hours after transfection, cells were depleted of sterols by direct addition of 2 ml of medium A containing 5% LPDS, 10 μM compactin, and 50 μM mevalonate (final concentrations) and incubated for 16 hr at 37°C. (B) Sterol-depleted cells, transfected with 10 ng pCMV-gp78-Myc (lanes 2–10; 12–20) and the indicated concentration of pCMV-Insig-1-T7, were switched to medium A containing 5% LPDS and 50 μM compactin in the absence (−) or presence (+) of sterols (1 μg/ml 25-HC and 10 μg/ml cholesterol). After incubation for 2 hr at 37°C, cells were harvested, lysed, and subjected to immunoprecipitation with anti-T7-coupled agarose beads. Aliquots of the pellet (5×) and supernatant (1×) fractions of immunoprecipitates were subjected to SDS-PAGE and transferred to nylon membranes. Immunoblot analysis was carried out with 1 μg/ml monoclonal anti-T7 IgG (against Insig-1) or 5 μg/ml monoclonal IgG-9E10 (against gp78). Filters were exposed to film at room temperature for 10 s. (C) Sterol-depleted cells, transfected with 10 ng pCMV-gp78-Myc, 20 ng pCMV-Insig-1-T7, and indicated concentration of pCIneo-gp78 (1–643) or pCIneo-gp78 (1–308) were harvested, lysed, and subjected to immunoprecipitation and immunoblot analysis as in (B). Filters were exposed to film at room temperature for 7 s. (D) Sterol-depleted cells, transfected with 20 ng pCMV-Insig-1-T7 and the indicated concentration of pCMV-gp78 (1–643)-Myc or pCMV-gp78 (1–308)-Myc, were harvested, lysed, and subjected to immunoprecipitation with anti-Myc beads. Immunoblot analysis was carried out as in (B). Filters were exposed to film at room temperature for 3 s. (E) Sterol-depleted cells, transfected with pCMV-Insig-1-Myc and the indicated concentration of pCMV-HA-Hrd1 and pCMV-HA-gp78 (1–643), were harvested, lysed, and subjected to immunoprecipitation with anti-Myc beads. Immunoblot analysis was carried out with 5 μg/ml monoclonal IgG-9E10 (against Insig-1) or 1 μg/ml monoclonal anti-HA IgG (against gp78 and Hrd1). Filters were exposed to film at room temperature for 1–5 s.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
The Membrane Domain of gp78 Inhibits Insig-Mediated Degradation of HMG CoA Reductase
(A–D) CHO-K1 cells were set up for experiments, transfected, and subsequently depleted of sterols as described in the legend to Figure 2. (A and B) Sterol-depleted cells, transfected with 1 μg pCMV-HMG-Red-T7, 30 ng pCMV-Insig-1-Myc or 100 ng pCMV-Insig-2-Myc, 100 ng pCIneo-gp78 (1–643) or pCIneo-gp78 (1–308) were switched to medium A containing 5% LPDS and 50 μM compactin in the absence (−) or presence (+) of 1 μg/ml 25-HC or lanosterol plus 10 mM mevalonate. Following incubation for 5 hr at 37°C, cells were harvested, membrane fractions were prepared, and aliquots (20–40 μg protein/lane) were subjected to SDS-PAGE. Immunoblot analysis was carried out with 1 μg/ml monoclonal anti-T7 IgG (against reductase) and 5 μg/ml IgG-9E10 (against Insig-1). (C) Sterol-depleted cells, transfected with 1 μg pCMV-HMG-Red-T7, 30 ng pCMV-Insig-1-Myc, 100 ng pCIneo-gp78 (1–308), and the indicated concentration of pCIneo-gp78 (1–643), were switched to medium A containing 5% LPDS and 50 μM compactin in the absence (−) or presence (+) of 1 μg/ml 25-HC plus 10 mM mevalonate. Following incubation for 5 hr at 37°C, cells were harvested, membrane fractions were prepared, and aliquots (15 μg protein/lane) were subjected to SDS-PAGE. Immunoblot analysis was carried out with 1 μg/ml monoclonal anti-T7 IgG (against reductase) and 5 μg/ml IgG-9E10 (against Insig-1). Filters were exposed to film at room temperature for 1–10 s. (D) Sterol-depleted cells, transfected with 1 μg pCMV-HMG-Red-T7, 30 ng pCMV-Insig-1-Myc, and the indicated concentration of pCMV-HA-gp78 (1–643) or pCMV-HA-gp78 (1–308), were switched to medium A containing 5% LPDS and 50 μM compactin in the absence (−) or presence (+) of 1 μg/ml 25-HC plus 10 mM mevalonate. Following incubation for 5 hr at 37°C, the cells were harvested, membrane fractions were prepared, and aliquots (20 μg protein/lane) were subjected to SDS-PAGE. Immunoblot analysis was carried out with 1 μg/ml monoclonal anti-T7 IgG (against reductase), 5 μg/ml IgG-9E10 (against Insig-1), or 1 μg/ml monoclonal anti-HA IgG (against gp78). Filters were exposed to film at room temperature for 1–10 s.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Insig-1 Mediates Formation of Sterol-Dependent Complex between Itself, HMG CoA Reductase, gp78, and VCP
(A and B) CHO-K1 cells were set up for experiments, transfected, and subsequently depleted of sterols as described in the legend to Figure 2. (A) Following sterol depletion, the cells, transfected with 1 μg of pCMV-HMG-Red-T7 (TM1-8), 20 ng pCMV-gp78-Myc, and 10 ng of pCMV-Insig-1-Myc, were switched to medium A containing 5% LPDS and 50 μM compactin in the absence (−) or presence (+) of 1 μg/ml 25-HC plus 10 mM mevalonate. Following incubation for 20 min at 37°C, cells were harvested, lysed, and subjected to immunoprecipitation with anti-T7 beads as described in Figure 2B. Immunoblot analysis was carried out with 1 μg/ml monoclonal anti-T7 IgG (against reductase) and 5 μg/ml monoclonal IgG-9E10 (against gp78 and Insig-1). Filters were exposed to film at room temperature for 5–60 s. (B) After sterol depletion, the cells, transfected with 1 μg of pCMV-Insig-1-T7 and 30 ng pCMV-gp78-Myc as indicated, were harvested, lysed, and subjected to immunoprecipitation as in (A). Equal proportions of the supernatant and pellet fractions of the immunoprecipitates were subjected to SDS-PAGE and immunoblotted with 1 μg/ml monoclonal anti-VCP, 1 μg/ml monoclonal anti-T7 IgG (against Insig-1), or 5 μg/ml monoclonal IgG-9E10 (against gp78). Filters were exposed to film at room temperature for 30–60 s.
(C) Stock cultures of SRD-13A cells were set up for experiments on day 0 at 7 × 105 cells per 60 mm dish. On day 1, the cells were transfected with 1 μg pCMV-HMG-Red-T7 (K89R/K248R), 1 μg pCMV-SCAP, 10 ng pCMV-Insig-1-T7, and 10 ng pCMV-gp78-Myc as indicated. Following sterol depletion, cells were incubated in the absence (−) or presence (+) of 1 μg/ml 25-HC plus 10 mM mevalonate. After 4 hr at 37°C, the cells were harvested, lysed, and subjected to immunoprecipitation as in Figure 2D. Immunoblot analysis was carried out with 5 μg/ml monoclonal IgG-9E10 (against gp78), 1 μg/ml monoclonal anti-T7 IgG (against Insig-1 and reductase), or 5 μg/ml polyclonal IgG-R139 (against SCAP). Filters were exposed to film at room temperature for 5–60s.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Gp78 and VCP Are Required for Sterol-Regulated Ubiquitination and Degradation of Endogenous HMG CoA Reductase as Revealed by RNA Interference
Stock cultures of SV-589 cells were set up on day 0 at 5 × 104 cells per 60 mm dish. On days 1 and 3, cells were transfected with indicated siRNA (400 pmol/dish) as described in Experimental Procedures. After the second transfection on day 3, the cells were depleted of sterols by incubating them for 16 hr at 37°C in medium B containing 10% LPDS, 50 μM compactin, and 50 μM mevalonate.
(A–F) Sterol-depleted cells were switched to medium B containing 10% LPDS, 50 μM compactin, and the indicated concentration of 25-HC. In (A), (C), and (E), the medium was also supplemented with 10 μM MG-132. Following incubation for 30 min at 37°C, the cells were harvested for immunoprecipitation (A, C, and E) or preparation of total RNA (B, D, and F). (A, C, and E) Cells were lysed and subjected to immunoprecipitation with polyclonal anti-reductase as described in Experimental Procedures. Aliquots of the immunoprecipitates were subjected to SDS-PAGE and immunoblotted with 5 μg/ml monoclonal IgG-A9 (against reductase) or 0.2 μg/ml monoclonal IgG-P4D1 (against ubiquitin). Filters were exposed to film at room temperature for 30 s to 1 min. (B, D, and F) Total RNA was prepared from cells treated with the highest concentration of sterols and subjected to first-strand cDNA synthesis and real-time PCR. Each value for cells transfected with the indicated siRNA represents the amount of the indicated mRNA relative to that in control cells transfected with VSV-G.
(G and H) Sterol-depleted cells were switched to medium B containing 10% LPDS, 50 μM compactin, and the indicated concentration of 25-HC or lanosterol plus 10 mM mevalonate. After 5 hr, cells were harvested, membrane fractions were prepared, and aliquots (25 μg protein/lane) were subjected to SDS-PAGE and immunoblotted with 5 μg/ml monoclonal IgG-A9 (against reductase), 1 μg/ml monoclonal anti-VCP IgG, and 1 μg/ml monoclonal anti-tranferrin receptor IgG. Filters were exposed to film at room temperature for 30 s.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Schematic Representation for the Pathway of Sterol-Accelerated Degradation of HMG CoA Reductase
The accumulation of sterols in ER membranes results in the exposure of an Insig binding site in the membrane domain of HMG CoA reductase. Binding of Insigs, along with its associated proteins gp78 and VCP, triggers the polyubiquitination of reductase in a reaction mediated by gp78 and its cognate E2. Ubiquitination of reductase marks the enzyme for recognition by VCP and its cofactors, which act to extract ubiquitinated reductase molecules from the ER membrane and present them to cytosolic proteasomes for degradation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions