Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Abstract

Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.

Keywords: Cholesterol Metabolism; Cytosolic Dislocation; ER-associated Degradation; Endoplasmic Reticulum (ER); Endoplasmic Reticulum-associated Protein Degradation (ERAD); Membrane Extraction; Membrane Trafficking; Proteasome.

FIGURE 1.

Peripheral association of HMG-CoA reductase with membranes of sterol-treated cells. UT-2/pHMG-Red-T7 cells were set up for experiments on day 0 in medium C at a density of 2 × 10⁵ (A) or 5 × 10⁵ (B and C) cells/100-mm dish. On day 3, the cells were switched to medium A supplemented with 5% LPDS, 10 μm compactin, 50 μm mevalonate, and 1 μm MG-132 in the absence or presence of 1 μg/ml 25-HC; 10 mm mevalonate (Mev.; A); 1, 3, or 10 mm mevalonate (B); and 1, 3, or 10 μm geranylgeraniol (GGOH; C) as indicated. Following incubation for 16 h at 37 °C, cells were harvested, resuspended in buffer containing 20 mm Tris-HCl (pH 7.4) and 250 mm sucrose, and lysed. Resulting lysates were then subjected to centrifugation at 1000 × g to generate postnuclear supernatants that were subjected to an additional round of centrifugation at 20,000 × g. Membrane pellets were resuspended in an equal volume of one of the following buffers: 20 mm Tris-HCl (pH 7.4) and 140 mm NaCl (Control), 20 mm Tris-HCl (pH 7.4) and 1 m KCl, 100 mm Na₂CO₃, and 20 mm Tris-HCl (pH 7.4) and 1% Nonidet P-40 (NP-40). Following rotation for 2 h at 4 °C, samples were layered on buffer containing 20 mm Tris-HCl (pH 7.4) and 500 mm sucrose and subjected to centrifugation for 30 min at 100,000 × g. Equal proportions of the resulting pellet (P) and supernatant (S) fractions were subjected to SDS-PAGE followed by immunoblot analysis with anti-T7 IgG (against reductase), MLO7 (against GM130), and anti-calnexin IgG.

FIGURE 2.

Sterol and nonsterol isoprenoids enhance susceptibility of lumenal T7 epitope in HMG-Red-T7 to trypsinolysis. A, topology of HMG-CoA reductase, Scap, and calnexin denoting the location of two T7 epitope tags in reductase encoded by pCMV-HMG-Red-T7, the epitope in Scap recognized by monoclonal IgG-9D5, and the region in calnexin recognized by anti-calnexin IgG. Sites for N-linked glycosylation in HMG-CoA reductase and Scap are indicated. B and C, UT-2/pHMG-Red-T7 cells were set up for experiments on day 0 at a density of 5 × 10⁵ cells/100-mm dish in medium C. On day 2, the cells were refed medium B supplemented with 5% LPDS, 10 μm compactin, 50 μm mevalonate (Mev.), and 1 μm MG-132 in the absence or presence of 1 μg/ml 25-HC plus 10 mm mevalonate as indicated. Following incubation at 37 °C for 16 h, the cells were harvested for subcellular fractionation using Protocol 1 as described under “Experimental Procedures.” The resulting membrane fractions were resuspended in Buffer B and digested with 2–20 μg of trypsin for 30 min at 30 °C (B) or with 20 μg of trypsin for the indicated amount of time at 30 °C (C). Following treatments, reactions were terminated, and the samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-T7 IgG (against reductase), IgG-9D5 (against Scap), and anti-calnexin IgG.

FIGURE 3.

Sterol and nonsterol requirements for extraction of HMG-CoA reductase across ER membranes as determined by protease protection. UT-2/pHMG-Red-T7 cells were set up for experiments on day 0 as described in the legend for Fig. 2B. On day 2, cells were refed medium A supplemented with 5% LPDS, 10 μm compactin, 50 μm mevalonate, and 1 μm MG-132 in the absence or presence of 1 μg/ml 25-HC plus the indicated concentration of mevalonate (A and C), 0–10 μm geranylgeraniol (GGOH; B and D), or 3 mm mevalonate plus either 1 μg/ml 25-HC or the indicated concentration of apomine (E). A, B, and E, following incubation at 37 °C for 16 h, the cells were harvested for subcellular fractionation using Protocol 1. The resulting membrane fractions were resuspended in Buffer B and pooled where appropriate, and equal amounts of membrane suspensions were digested with 10–20 μg of trypsin for 30 min at 30 °C. Reactions were then terminated, and samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-T7 IgG (against reductase), IgG-9D5 (against Scap), and anti-calnexin IgG. C and D, following incubation for 16 h at 37 °C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (10 μg of protein/lane) were subjected to SDS-PAGE followed by immunoblot analysis with anti-T7 IgG (against reductase) and anti-calnexin IgG. Asterisks denote non-specific bands.

FIGURE 4.

RNAi-mediated knockdown of Insigs or VCP/p97 blunts sterol-induced extraction of HMG-CoA reductase across ER membranes. UT-2/pHMG-Red-T7 cells were set up on day 0 at a density of 1.5–2 × 10⁵ cells/60-mm dish in medium C. On days 1 and 2, the cells were transfected with the indicated siRNA duplexes as described under “Experimental Procedures.” Following transfection on day 2, cells received a direct addition of medium A containing 5% LPDS, 10 μm compactin, and 50 μm mevalonate (Mev.) in the absence or presence of 1 μg/ml 25-HC plus 10 mm mevalonate (final concentrations). In B and D, some of the cells (lanes 1–4) were also treated with 1 μm MG-132; all of the cells in E were treated with 1 μm MG-132. Following incubation at 37 °C for 16 h, cells were harvested for subcellular fractionation using Protocol 1(A and B) or Protocol 2 (C and D). Aliquots of the resulting membrane fractions were resuspended in Buffer B and pooled where appropriate, and equal amounts of membrane suspensions were digested with 10 μg of trypsin. Some of the samples (lanes 5, 6, 11, and 12 in A) were treated with 1% Nonidet P-40 (NP-40) prior to proteolysis. After 30 min at 30 °C, the reactions were terminated, and samples were subjected to SDS-PAGE followed by immunoblot analysis with anti-T7 IgG (against reductase), IgG-9D5 (against Scap), IgG-18 (against VCP/p97), and anti-calnexin IgG. Asterisks denote non-specific bands.

FIGURE 5.

RNAi-mediated knockdown of AAA-ATPases of the proteasome 19 S regulatory particle blunts sterol-accelerated degradation and cytosolic dislocation of HMG-CoA reductase. CHO-K1 cells were set up on day 0 at a density of 5 × 10⁵ cells/100-mm dish (A and C) or 1.5 × 10⁵ cells/60-mm dish (B) in medium A containing 5% FCS. On day 1, cells were transfected with the indicated siRNA duplexes in A, Rpt5-1 in B, and Rpt4-3 or Rpt5-1 in C as described in the legend for Fig. 4. Four hours following transfection, cells were depleted of sterols for 16 h by the direct addition of medium A containing 5% LPDS, 10 μm compactin, and 50 μm mevalonate (Mev.) (final concentrations). A, sterol-depleted cells were switched to identical medium in the absence or presence of 1 μg/ml 25-HC plus 10 mm mevalonate as indicated. After 4–5 h at 37 °C, cells were harvested and subjected to cellular fractionation. Aliquots of resulting membrane fractions (15–20 μg of protein/lane) were subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against reductase) and anti-calnexin IgG. B and C, sterol-depleted cells were pretreated for 1 h with medium A supplemented with 5% LPDS, 10 μm compactin, and 50 μm mevalonate in the absence or presence of 10 μm MG-132. Cells then received medium A containing 5% LPDS, 10 μm compactin, and 50 μm mevalonate in the absence or presence of 10 μm MG-132 and 1 μg/ml 25-HC plus 10 mm mevalonate. After 4 h at 37 °C, cells were harvested, and postnuclear supernatants were subjected to centrifugation at 1 × 10⁶ g for 30 min at 4 °C. Aliquots of the resulting membrane (Memb.) pellet (15–20 μg of protein/lane) and cytosolic supernatant (10–40 μg of protein/lane) fractions were subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against reductase), IgG-TBP1-19 (against Rpt5), IgG-p42-23 (against Rpt4), and anti-actin IgG. Bands corresponding to cytosolically dislocated reductase were quantified using ImageJ software. The intensities of these bands in the absence of 25-HC plus mevalonate were arbitrarily set as 1 for each of the RNAi-mediated knockdowns. Results are representative of at least three independent experiments.

FIGURE 6.

Sterol and nonsterol isoprenoids trigger membrane extraction of HMG-CoA reductase in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. UT-2/pHMG-Red-T7 (A and B) and CHO-K1 (C and D) cells were set up for experiments, transfected with the Rpt5-1 or Rpt2-1 siRNA duplex as indicated, and treated in the absence or presence of 1 μm MG-132, 1 μg/ml 25-HC, and 10 mm mevalonate (Mev.) as described in the legends for Figs. 4 and 5. A and B, following treatments, cells were harvested for subcellular fractionation using Protocol 1, and aliquots of resulting membranes were subjected to trypsinolysis as described in the legends for Figs. 4 and 5. After trypsinolysis, samples together with aliquots of the cytosol were subjected to SDS-PAGE, and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-18 (against VCP/p97), IgG-TBP1-19 (against Rpt5), anti-Rpt2 IgG, and anti-calnexin IgG. Bands corresponding to the protected fragments of reductase in the anti-T7 immunoblots were quantified using ImageJ software. The intensities of the protected fragments of reductase in the absence of 25-HC plus mevalonate were arbitrarily set as 1 for each of the RNAi-mediated knockdowns. C, membranes from control and Rpt5 knockdown cells treated with 25-HC plus 10 mm mevalonate and 10 μm MG-132 were resuspended in TBS containing 71.5% sucrose, and the samples were then overlaid with TBS containing 65 and 10% sucrose. After centrifugation at 100,000 × g for 16 h at 4 °C, aliquots were removed from the top to the bottom of the gradient and analyzed by SDS-PAGE and immunoblot analysis with anti-T7 IgG (against reductase), anti-calnexin IgG, and IgG-TBP1-19 (against Rpt5). D, membranes from control and Rpt5 knockdown cells treated with 10 μm MG-132, 1 μg/ml 25-HC, and 10 mm mevalonate were resuspended in TBS with or without 0.1% Nonidet P-40 (NP-40). Following rotation at 4 °C for 1 h, the samples were separated into soluble supernatant and insoluble pellet fractions by centrifugation. Aliquots of the fractions were then analyzed by immunoblotting with IgG-A9 (against reductase) and anti-calnexin IgG.

FIGURE 7.

Model for the Insig-mediated, sterol-accelerated ER-associated degradation of HMG-CoA reductase. The diagram shows a schematic representation of the sterol-accelerated ERAD of HMG-CoA reductase. Sterol-induced binding to Insigs results in ubiquitination of HMG-CoA reductase on two cytosolic lysine residues in the membrane domain. Ubiquitination of reductase, which is mediated by two Insig-associated ubiquitin ligases called gp78 and Trc8, marks the enzyme for VCP/p97-mediated extraction across ER membranes through a reaction that is enhanced by geranylgeraniol. Partially extracted reductase with the T7-tagged lumenal loop between transmembrane helices 7 and 8 exposed to the cytosol is indicated as an intermediate in the complete extraction of reductase. Once extracted, reductase is released from membranes into the cytosol through a reaction mediated by the proteasome 19 S RP and finally delivered into the 20 S proteasome CP for degradation.