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

Elsabrouty R, Jo Y, Dinh TT, DeBose-Boyd RA - Mol. Biol. Cell (2013)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046.

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.

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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).
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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).

Mentions: In intact cells, GGOH augments sterol-accelerated degradation and cytosolic dislocation of reductase (Sever et al., 2003a). The experiment shown in Figure 6A was designed to determine whether GGOH also enhanced 25-HC–induced dislocation of reductase in permeabilized cells. In the absence of GGOH, 25-HC stimulated the ubiquitination of reductase in the pellet fraction (Figure 6A, panel 1, lane b); this was marginally enhanced by the addition of increasing concentrations of GGOH (lanes d, f, h, and j). Treatment of the permeabilized cells with 25-HC also stimulated dislocation of ubiquitinated, as well as intact, full-length reductase into the supernatant (panels 3 and 4, lane b), and the reaction was augmented by GGOH in a dose-dependent manner (panels 3 and 4, lanes d, f, h, and j).


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

Elsabrouty R, Jo Y, Dinh TT, DeBose-Boyd RA - Mol. Biol. Cell (2013)

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).
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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).
Mentions: In intact cells, GGOH augments sterol-accelerated degradation and cytosolic dislocation of reductase (Sever et al., 2003a). The experiment shown in Figure 6A was designed to determine whether GGOH also enhanced 25-HC–induced dislocation of reductase in permeabilized cells. In the absence of GGOH, 25-HC stimulated the ubiquitination of reductase in the pellet fraction (Figure 6A, panel 1, lane b); this was marginally enhanced by the addition of increasing concentrations of GGOH (lanes d, f, h, and j). Treatment of the permeabilized cells with 25-HC also stimulated dislocation of ubiquitinated, as well as intact, full-length reductase into the supernatant (panels 3 and 4, lane b), and the reaction was augmented by GGOH in a dose-dependent manner (panels 3 and 4, lanes d, f, h, and j).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046.

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.

Show MeSH
Related in: MedlinePlus