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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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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).
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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).

Mentions: The recombinant catalytic domain of ubiquitin specific protease 2 (USP2-cd), which efficiently removes ubiquitin from polyubiquitin chains attached to substrates (Ryu et al., 2006), was next used to demonstrate that sterols trigger dislocation of ubiquitinated reductase from membranes of permeabilized cells. In the experiment of Figure 4A, permeabilized cells were incubated with rat liver cytosol and ATP in the absence or presence of 25-HC to allow for the ubiquitination and subsequent dislocation of reductase. After 75 min, reactions were supplemented with various amounts of USP2-cd for an additional 15 min. The reactions were then terminated, and pellet and supernatant fractions of the permeabilized cells were analyzed sequentially by immunoprecipitation and immunoblot. The results show that, as expected, 25-HC stimulated ubiquitination of reductase in the pellet fraction of control reactions (Figure 4A, panel 1, compare lanes 1 and 2); this ubiquitination was reduced by USP2-cd in a concentration-dependent manner (lanes 4, 6, 8, and 10). Ubiquitinated forms of reductase appeared in the supernatant of 25-HC–treated permeabilized cells (panel 3, lane 12); this appearance was reduced by USP2-cd treatment (lanes 14, 16, 18, and 20). The amount of full-length reductase in the pellet fraction of permeabilized cells remained constant throughout the assay (Figure 4A, panel 2, lanes 1–10); however, treatment with USP2-cd before immunoprecipitation and immunoblot analysis markedly enhanced the amount of full-length reductase that was detected in the supernatant (panel 4, compare lane 12 with lanes 14, 16, 18, and 20).


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)

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).
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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).
Mentions: The recombinant catalytic domain of ubiquitin specific protease 2 (USP2-cd), which efficiently removes ubiquitin from polyubiquitin chains attached to substrates (Ryu et al., 2006), was next used to demonstrate that sterols trigger dislocation of ubiquitinated reductase from membranes of permeabilized cells. In the experiment of Figure 4A, permeabilized cells were incubated with rat liver cytosol and ATP in the absence or presence of 25-HC to allow for the ubiquitination and subsequent dislocation of reductase. After 75 min, reactions were supplemented with various amounts of USP2-cd for an additional 15 min. The reactions were then terminated, and pellet and supernatant fractions of the permeabilized cells were analyzed sequentially by immunoprecipitation and immunoblot. The results show that, as expected, 25-HC stimulated ubiquitination of reductase in the pellet fraction of control reactions (Figure 4A, panel 1, compare lanes 1 and 2); this ubiquitination was reduced by USP2-cd in a concentration-dependent manner (lanes 4, 6, 8, and 10). Ubiquitinated forms of reductase appeared in the supernatant of 25-HC–treated permeabilized cells (panel 3, lane 12); this appearance was reduced by USP2-cd treatment (lanes 14, 16, 18, and 20). The amount of full-length reductase in the pellet fraction of permeabilized cells remained constant throughout the assay (Figure 4A, panel 2, lanes 1–10); however, treatment with USP2-cd before immunoprecipitation and immunoblot analysis markedly enhanced the amount of full-length reductase that was detected in the supernatant (panel 4, compare lane 12 with lanes 14, 16, 18, and 20).

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