Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

Abstract

The polytopic endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.

FIGURE 1:

RNAi-mediated knockdown of VCP/p97 blocks sterol-accelerated degradation but not ubiquitination of HMG CoA reductase in SV-589 cells. SV-589 cells were set up on day 0 at 4 × 10⁵ cells/100-mm dish in medium A supplemented with 10% FCS. On days 1 and 2, cells were transfected in medium A containing 10% FCS with siRNAs targeting the control mRNA, VSV-G, or the VCP/p97 mRNA as indicated and described in Materials and Methods. After the second transfection on day 2, the cells were depleted of sterols through incubation for 16 h at 37°C in medium A supplemented with 10% lipoprotein-deficient serum (LPDS), 10 μM sodium compactin, and 50 μM sodium mevalonate. The cells were subsequently treated with medium A containing 10% LPDS and 10 μM compactin in the absence or presence of 10 μM MG-132 as indicated for 0.5 h at 37°C. The cells were then treated for an additional 0.5 (A, lanes 1–6; B, lanes 1–8) or 4 h (A, lanes 7–12; B, lanes 9–16) in the absence or presence of 1 μg/ml 25-HC plus 10 mM mevalonate (Mev.). At the end of the incubations, the cells were harvested, lysed in detergent-containing buffer, and subjected to immunoprecipitation with polyclonal anti-reductase as described in Materials and Methods. Aliquots of immunoprecipitates and total lysates were subjected to SDS–PAGE, and immunoblot analysis was carried out with IgG-P4D1 (against ubiquitin), IgG-A9 (against reductase), or anti-VCP/p97 IgG. Numbers next to immunoblots are referred to as “panels” in the text.

FIGURE 2:

Sterol-induced dislocation of HMG CoA reductase from membranes of permeabilized SV-589 cells. SV-589 cells were set up for experiments on day 0 at 2 × 10⁵ cells/100-mm dish in medium A supplemented with 10% FCS. On day 4, cells were washed with PBS and depleted of sterols through incubation in medium A containing 10% LPDS, 10 μM compactin, and 50 μM mevalonate for 16 h at 37°C. The sterol-depleted cells were subsequently harvested into the medium, washed with PBS containing 0.9 mM CaCl₂, and permeabilized with 0.025% digitonin as described in Materials and Methods. (A) Permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors (10 μM MG-132, 5 μg/ml pepstatin, and 2 μg/ml aprotinin) and 0.1 mg/ml FLAG-ubiquitin in the absence or presence of 10 μg/ml 25-HC, the ATP-regenerating system, and 2 mg/ml rat liver cytosol as indicated. After 75 min at 37°C, the reactions were terminated; the samples were homogenized in the absence of detergents, and resulting lysates were subjected to centrifugation at 100,000 × g for 30 min at 4°C. The pellet and supernatant fractions of this spin were then immunoprecipitated with polyclonal anti-HMG CoA reductase IgG as described in Materials and Methods. Aliquots of the immunoprecipitates were subjected to SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with IgG-A9 (against reductase) or IgG-M2 (against FLAG-ubiquitin). (B–D) Permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors, an ATP-regenerating system, and 0.1 mg/ml FLAG-ubiquitin. (B) Reactions received 2 mg/ml rat liver cytosol and were incubated in the absence or presence of 10 μg/ml 25-HC at 37°C. After the indicated period of time, reactions were terminated; the samples were lysed and separated into pellet and supernatant fractions by 100,000 × g centrifugation, followed by immunoprecipitation and immunoblot analysis as in A. (C) Rat liver cytosol was added to reactions at concentrations ranging from 0.1 to 3 mg/ml and incubated in the absence or presence of 10 μg/ml 25-HC as indicated. After incubation at 37°C for 75 min, samples were fractionated and subjected to immunoprecipitation and immunoblot as in A. (D) Reactions were incubated with 2 mg/ml rat liver cytosol and the indicated concentration of 25-HC. After incubation at 37°C for 75 min, samples were fractionated and subjected to immunoprecipitation and immunoblot as in A.

FIGURE 3:

RNAi-mediated knockdown of Insigs blunts sterol-induced dislocation of HMG CoA reductase from membranes of permeabilized SV-589 cells. SV-589 cells were set up on day 0 at 1 × 10⁵ cells/100-mm dish in medium A supplemented with 10% FCS. On day 3, the cells were transfected with siRNAs targeting the control mRNA, GFP, or mRNAs encoding Insig-1 and Insig-2 as indicated and depleted of sterols as described in the legend to Figure 1. After sterol depletion, the cells were harvested and permeabilized as described in the legend to Figure 2. Pellets of permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors, the ATP regenerating system, 0.1 mg/ml FLAG-ubiquitin, and 2 mg/ml rat liver cytosol in the absence or presence of 10 μg/ml 25-HC. After incubation for 90 min at 37°C, reactions were terminated, and samples were subjected sequentially to fractionation and anti-reductase immunoprecipitation; the resulting pellet and supernatant fractions, along with aliquots of the cell lysates, were analyzed by immunoblot with IgG-A9 (against reductase), IgG-M2 (against FLAG-ubiquitin), and IgG-17H1 (against Insig-1).

FIGURE 4:

Sterols trigger the dislocation of ubiquitinated forms of HMG CoA reductase from membranes of permeabilized SV-589 cells. (A–C) SV-589 cells were set up on day 0, depleted of sterols on day 4, and permeabilized with 0.025% digitonin as described in the legend to Figure 2. Pellets of permeabilized cells were then resuspended in permeabilization buffer containing protease inhibitors, the ATP-regenerating system, 0.1 mg/ml FLAG-ubiquitin, and 2 mg/ml rat liver cytosol in the absence or presence of 10 μg/ml 25-HC as indicated. Some of the reactions in B and C also received the indicated amount of ubiquitin aldehyde. After incubation for 75 min at 37°C, the reactions were supplemented with either increasing amounts of recombinant USP2-cd (A) or a constant amount of the enzyme (1.7 μg; B and C) and incubated for an additional 15 min at 37°C. Reactions were then terminated, and the samples were fractionated and subjected to anti-reductase immunoprecipitation, followed by immunoblot analysis with IgG-A9 (against reductase) or IgG-M2 (against FLAG-ubiquitin).

FIGURE 5:

The 1,1-bisphosphonate esters Apomine and SR-12813 stimulate ubiquitination and dislocation of HMG CoA reductase from membranes of permeabilized SV-589 cells. SV-589 cells were set up on day 0, depleted of sterols on day 4, and permeabilized with 0.025% digitonin as described in the legend to Figure 2. Pellets of permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors, the ATP-regenerating system, 0.1 mg/ml FLAG-ubiquitin, and 2 mg/ml rat liver cytosol. (A, B) Reactions were supplemented with the indicated concentration of Apomine (A) or SR-12813 (B). After incubation for 75 min at 37°C, 1.7 μg of USP2-cd was added, and reactions were incubated for an additional 15 min at 37°C. Reactions were then stopped, and samples were subjected sequentially to fractionation, immunoprecipitation, and immunoblot analysis with IgG-A9 (against reductase) or IgG-M2 (against FLAG-ubiquitin).

FIGURE 6:

The nonsterol isoprenoid geranylgeraniol augments sterol-induced dislocation of HMG CoA reductase from membranes of permeabilized SV-589 cells. (A–C) SV-589 cells were set up on day 0 and depleted of sterols on day 4 as described in the legend to Figure 2. (A) Sterol-depleted cells were harvested and permeabilized with 0.025% digitonin as described in the legend to Figure 2. The permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors, the ATP regeneration system, 0.1 mg/ml FLAG-ubiquitin, and 2 mg/ml rat liver cytosol in the absence or presence of 10 μg/ml 25-HC and the indicated concentration of GGOH. After incubation for 75 min at 37°C, 1.7 μg of USP2-cd was added and reactions were incubated for an additional 15 min at 37°C. Reactions were then stopped, and samples were subjected sequentially to fractionation, immunoprecipitation, and immunoblot analysis with IgG-A9 (against reductase) or IgG-M2 (against FLAG-ubiquitin). (B, C) Sterol-depleted cells were pretreated for 1 h at 37°C in medium A containing 10% LPDS, 10 μM compactin, 50 μM mevalonate, and 10 μM MG-132; the cells were subsequently switched to identical medium in the absence or presence of 1 μg/ml 25-HC. After 2 h at 37°C, the cells were harvested and permeabilized with 0.025% digitonin as described in the legend to Figure 2. The permeabilized cells were resuspended in permeabilization buffer containing protease inhibitors, the ATP regeneration system, 2 mg/ml rat liver cytosol, and GGOH or FOH as indicated. The amount of GGOH used in C was 15 μM. After incubation for 75 min at 37°C, 1.7 μg of USP2-cd was added, and reactions were incubated for an additional 15 min at 37°C. Reactions were then stopped, and samples were subjected sequentially to fractionation, immunoprecipitation, and immunoblot analysis with IgG-A9 (against reductase) and IgG-P4D1 (against ubiquitin).