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The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schumacher MM, Elsabrouty R, Seemann J, Jo Y, DeBose-Boyd RA - Elife (2015)

Bottom Line: Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

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Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.DOI:http://dx.doi.org/10.7554/eLife.05560.011
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fig5s3: Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.DOI:http://dx.doi.org/10.7554/eLife.05560.011

Mentions: Consistent with a role in the ERAD of reductase, UBIAD1 has been localized to membranes of the ER (Nakagawa et al., 2010; Nickerson et al., 2013). However, it should be noted that the prenyltransferase has also been localized to membranes of the mitochondria (Nickerson et al., 2010) as well as the Golgi (Mugoni et al., 2013; Wang et al., 2013). In the experiment shown in Figure 5C, we examined the subcellular localization of endogenous UBIAD1 in SV-589 cells grown in medium containing FCS (fetal calf serum). The results show that endogenous UBIAD1 localized to juxtanuclear structures that resembled the Golgi and also stained with antibodies against the Golgi protein GM130 (Nakamura et al., 1995) (Figure 5C, panels 1 and 2). The Golgi localization of UBIAD1 was markedly diminished when the cells were depleted of sterol and nonsterol isoprenoids through incubation in medium containing LPPS (lipoprotein-poor serum) and the reductase inhibitor compactin (Figure 5C, panels 3 and 4). Notably, localization of Golgi-localized GM130 was unaffected by depletion of sterol and nonsterol isoprenoids (compare panels 2 and 4). The Golgi localization of UBIAD1 was restored in sterol- and nonsterol isoprenoid-depleted cells by the addition of geranylgeraniol (Figure 5C, panel 5), but not by the addition of 25-HC (panel 7). A repeat experiment confirms that sterol and nonsterol isoprenoid depletion disrupted the Golgi localization of UBIAD1 (Figure 5—figure supplement 2). Importantly, this localization was restored by geranylgeraniol, but not by farnesol, 25-HC, or cholesterol. We obtained similar results with SV-589 cells stably transfected with pCMV-Myc-UBIAD1 encoding Myc-tagged human UBIAD1. Depletion of sterol and nonsterol isoprenoids disrupted Golgi localization of transfected Myc-UBIAD1 (Figure 5—figure supplement 3). This localization was restored by geranylgeraniol, but not by farnesol or 25-HC. Notably, endogenous UBIAD1 exhibited relatively more non-Golgi staining compared to Myc-UBIAD1 in FCS-cultured cells, suggesting anti-Myc may have a greater degree of specificity compared to that of anti-UBIAD1.


The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schumacher MM, Elsabrouty R, Seemann J, Jo Y, DeBose-Boyd RA - Elife (2015)

Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.DOI:http://dx.doi.org/10.7554/eLife.05560.011
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fig5s3: Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.DOI:http://dx.doi.org/10.7554/eLife.05560.011
Mentions: Consistent with a role in the ERAD of reductase, UBIAD1 has been localized to membranes of the ER (Nakagawa et al., 2010; Nickerson et al., 2013). However, it should be noted that the prenyltransferase has also been localized to membranes of the mitochondria (Nickerson et al., 2010) as well as the Golgi (Mugoni et al., 2013; Wang et al., 2013). In the experiment shown in Figure 5C, we examined the subcellular localization of endogenous UBIAD1 in SV-589 cells grown in medium containing FCS (fetal calf serum). The results show that endogenous UBIAD1 localized to juxtanuclear structures that resembled the Golgi and also stained with antibodies against the Golgi protein GM130 (Nakamura et al., 1995) (Figure 5C, panels 1 and 2). The Golgi localization of UBIAD1 was markedly diminished when the cells were depleted of sterol and nonsterol isoprenoids through incubation in medium containing LPPS (lipoprotein-poor serum) and the reductase inhibitor compactin (Figure 5C, panels 3 and 4). Notably, localization of Golgi-localized GM130 was unaffected by depletion of sterol and nonsterol isoprenoids (compare panels 2 and 4). The Golgi localization of UBIAD1 was restored in sterol- and nonsterol isoprenoid-depleted cells by the addition of geranylgeraniol (Figure 5C, panel 5), but not by the addition of 25-HC (panel 7). A repeat experiment confirms that sterol and nonsterol isoprenoid depletion disrupted the Golgi localization of UBIAD1 (Figure 5—figure supplement 2). Importantly, this localization was restored by geranylgeraniol, but not by farnesol, 25-HC, or cholesterol. We obtained similar results with SV-589 cells stably transfected with pCMV-Myc-UBIAD1 encoding Myc-tagged human UBIAD1. Depletion of sterol and nonsterol isoprenoids disrupted Golgi localization of transfected Myc-UBIAD1 (Figure 5—figure supplement 3). This localization was restored by geranylgeraniol, but not by farnesol or 25-HC. Notably, endogenous UBIAD1 exhibited relatively more non-Golgi staining compared to Myc-UBIAD1 in FCS-cultured cells, suggesting anti-Myc may have a greater degree of specificity compared to that of anti-UBIAD1.

Bottom Line: Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

Show MeSH
Related in: MedlinePlus