Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814148&req=5

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.

Mentions: In the experiment of Figure 2A, permeabilized SV-589 cells were subjected to in vitro treatments with FLAG-tagged ubiquitin in the absence or presence of the ATP-regenerating system, rat liver cytosol, and 25-HC. After incubation at 37°C, the cells were homogenized in the absence of detergents and subjected to 100,000 × g centrifugation. The pellet and supernatant fractions of this spin were mixed with detergent-containing buffer and immunoprecipitated with anti-reductase polyclonal antibodies. The precipitated material from both fractions was then analyzed by immunoblot with anti-FLAG (Figure 2A, panels 1 and 3) or anti-reductase (panels 2 and 4) monoclonal antibodies. Incubation of permeabilized cells with 25-HC led to the ubiquitination of reductase in the 100,000 × g pellet fraction when reactions were also supplemented with rat liver cytosol and the ATP-regenerating system (Figure 2A, panel 1, compare lanes 2, 4, and 6 with lane 8). The amount of total reductase immunoprecipitated from the pellet fractions remained constant throughout the assay (panel 2, lanes 1–8). Immunoblot analysis of reductase immunoprecipitates from the 100,000 × g supernatant fractions revealed that ubiquitinated, as well as intact, full-length forms of reductase became dislocated from membranes of permeabilized cells but only when reactions were supplemented with 25-HC, the ATP-regenerating system, and rat liver cytosol (Figure 2A, panels 3 and 4, lane 8). Figure 2B shows that the amount of ubiquitinated reductase in the pellet fraction of 25-HC–treated permeabilized cells rose with time, reaching a plateau after 30 min (panel 1, lanes 3–10). Dislocation of ubiquitinated and full-length reductase was observed after 30 min of incubation with 25-HC (Figure 2B, panels 3 and 4, lane 6), and this reached a maximum after 90 min (panels 3 and 4, lane 10). Finally, reductase ubiquitination and dislocation in permeabilized cells was proportional to the amount of rat liver cytosol in reactions (Figure 2C, panels 1–4, lanes 8 and 10) and 25-HC (Figure 2D, panels 1–4, lanes 2–6). Note that Insig-1 appears to become dislocated into the cytosol along with reductase (Leichner et al., 2009). We were unable to detect Insig-1 in the supernatant fractions of permeabilized cells due to insufficient sensitivity of anti–Insig-1 antibodies. However, experiments of Supplemental Figure S3 show that permeabilized SV-589 cells support dislocation of overexpressed Insig-1 in a manner dependent on ATP, rat liver cytosol, and time of incubation.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814148&req=5

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.
Mentions: In the experiment of Figure 2A, permeabilized SV-589 cells were subjected to in vitro treatments with FLAG-tagged ubiquitin in the absence or presence of the ATP-regenerating system, rat liver cytosol, and 25-HC. After incubation at 37°C, the cells were homogenized in the absence of detergents and subjected to 100,000 × g centrifugation. The pellet and supernatant fractions of this spin were mixed with detergent-containing buffer and immunoprecipitated with anti-reductase polyclonal antibodies. The precipitated material from both fractions was then analyzed by immunoblot with anti-FLAG (Figure 2A, panels 1 and 3) or anti-reductase (panels 2 and 4) monoclonal antibodies. Incubation of permeabilized cells with 25-HC led to the ubiquitination of reductase in the 100,000 × g pellet fraction when reactions were also supplemented with rat liver cytosol and the ATP-regenerating system (Figure 2A, panel 1, compare lanes 2, 4, and 6 with lane 8). The amount of total reductase immunoprecipitated from the pellet fractions remained constant throughout the assay (panel 2, lanes 1–8). Immunoblot analysis of reductase immunoprecipitates from the 100,000 × g supernatant fractions revealed that ubiquitinated, as well as intact, full-length forms of reductase became dislocated from membranes of permeabilized cells but only when reactions were supplemented with 25-HC, the ATP-regenerating system, and rat liver cytosol (Figure 2A, panels 3 and 4, lane 8). Figure 2B shows that the amount of ubiquitinated reductase in the pellet fraction of 25-HC–treated permeabilized cells rose with time, reaching a plateau after 30 min (panel 1, lanes 3–10). Dislocation of ubiquitinated and full-length reductase was observed after 30 min of incubation with 25-HC (Figure 2B, panels 3 and 4, lane 6), and this reached a maximum after 90 min (panels 3 and 4, lane 10). Finally, reductase ubiquitination and dislocation in permeabilized cells was proportional to the amount of rat liver cytosol in reactions (Figure 2C, panels 1–4, lanes 8 and 10) and 25-HC (Figure 2D, panels 1–4, lanes 2–6). Note that Insig-1 appears to become dislocated into the cytosol along with reductase (Leichner et al., 2009). We were unable to detect Insig-1 in the supernatant fractions of permeabilized cells due to insufficient sensitivity of anti–Insig-1 antibodies. However, experiments of Supplemental Figure S3 show that permeabilized SV-589 cells support dislocation of overexpressed Insig-1 in a manner dependent on ATP, rat liver cytosol, and time of incubation.

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