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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 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).
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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).

Mentions: In cultured cells, the 1,1-bisphosphonate esters Apomine and SR-12813 mimic 25-HC in stimulating Insig-mediated ubiquitination and subsequent degradation of reductase in intact cells (Roitelman et al., 2004; Sever et al., 2004). Having optimized the assay for sterol-induced dislocation of reductase in permeabilized cells using either ubiquitin aldehyde or USP2-cd (Figure 4), we next designed experiments to determine whether SR-12813 and Apomine trigger the reaction. The results show that, in a dose-dependent manner, both Apomine and SR-12813 stimulated ubiquitination of reductase in the pellet fraction of permeabilized cells (Figure 5, A and B, panel 1, compare lane a with lanes b–g). The bisphosphonate esters also triggered the dislocation of ubiquitinated and full-length forms of the enzyme into the supernatant fraction (Figure 5, A and B, panels 3 and 4, lanes b–g).


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

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

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).
Mentions: In cultured cells, the 1,1-bisphosphonate esters Apomine and SR-12813 mimic 25-HC in stimulating Insig-mediated ubiquitination and subsequent degradation of reductase in intact cells (Roitelman et al., 2004; Sever et al., 2004). Having optimized the assay for sterol-induced dislocation of reductase in permeabilized cells using either ubiquitin aldehyde or USP2-cd (Figure 4), we next designed experiments to determine whether SR-12813 and Apomine trigger the reaction. The results show that, in a dose-dependent manner, both Apomine and SR-12813 stimulated ubiquitination of reductase in the pellet fraction of permeabilized cells (Figure 5, A and B, panel 1, compare lane a with lanes b–g). The bisphosphonate esters also triggered the dislocation of ubiquitinated and full-length forms of the enzyme into the supernatant fraction (Figure 5, A and B, panels 3 and 4, lanes b–g).

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