UbiA prenyltransferase domain-containing protein-1 modulates HMG-CoA reductase degradation to coordinate synthesis of sterol and nonsterol isoprenoids

Abstract

UBIAD1 (UbiA prenyltransferase domain-containing protein-1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K₂ We previously reported that sterols stimulate binding of UBIAD1 to endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. UBIAD1 binding inhibits sterol-accelerated, ER-associated degradation (ERAD) of reductase, one of several mechanisms for feedback control of this rate-limiting enzyme in the branched pathway that produces cholesterol and nonsterol isoprenoids such as GGpp. Accumulation of GGpp in ER membranes triggers release of UBIAD1 from reductase, permitting its maximal ERAD and ER-to-Golgi transport of UBIAD1. Mutant UBIAD1 variants associated with Schnyder corneal dystrophy (SCD), a human disorder characterized by corneal accumulation of cholesterol, resist GGpp-induced release from reductase and remain sequestered in the ER to block reductase ERAD. Using lines of genetically manipulated cells, we now examine consequences of UBIAD1 deficiency and SCD-associated UBIAD1 on reductase ERAD and cholesterol synthesis. Our results indicated that reductase becomes destabilized in the absence of UBIAD1, resulting in reduced cholesterol synthesis and intracellular accumulation. In contrast, an SCD-associated UBIAD1 variant inhibited reductase ERAD, thereby stabilizing the enzyme and contributing to enhanced synthesis and intracellular accumulation of cholesterol. Finally, we present evidence that GGpp-regulated, ER-to-Golgi transport enables UBIAD1 to modulate reductase ERAD such that synthesis of nonsterol isoprenoids is maintained in sterol-replete cells. These findings further establish UBIAD1 as a central player in the reductase ERAD pathway and regulation of isoprenoid synthesis. They also indicate that UBIAD1-mediated inhibition of reductase ERAD underlies cholesterol accumulation associated with SCD.

Keywords: Golgi; cholesterol metabolism; endoplasmic reticulum (ER); endoplasmic reticulum–associated protein degradation (ERAD); isoprenoid.

Figure 1.

Expression of HMG CoA reductase protein is reduced in absence of UBIAD1 and enhanced in presence of SCD-associated UBIAD1 (N102S). A and B, SV-589, SV-589(ΔUBIAD1), SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT), and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 4 × 10⁵ cells/60-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were refed medium A supplemented with either 10% FCS or LPDS as indicated. After 16 h at 37 °C, the cells were harvested for subcellular fractionation (A) or preparation of total RNA (B) as described under “Experimental procedures.” A, aliquots of membrane fractions (20 μg protein/lane) were subjected to SDS-PAGE, and immunoblot analysis was carried with IgG-A9 (against HMG CoA reductase), IgG-H8 (against endogenous UBIAD1), IgG-D1 (against ACAT-1), and anti-calnexin IgG. B, total RNA was subjected to quantitative RT-PCR using primers against human reductase; the human 36B4 mRNA was used as an invariant control. Each value represents the amount of reductase mRNA relative to that in SV-589 cells, which is arbitrarily defined as 1. C, SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 3 × 10⁵ cells/60-mm dish in medium A containing 10% FCS. On day 1, the cells were refed identical medium and incubated for 16 h at 37 °C. The cells were then fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) using a Zeiss Axio Observer Epifluorescence microscope as described under “Experimental procedures.” D, SV-589, SV-589(ΔUBIAD1), SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT), and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 5 × 10⁵ cells/100-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were depleted of sterols through incubation in medium A supplemented with 10% LPDS, 10 μm compactin, and 50 μm mevalonate. After 16 h at 37 °C, the cells were refed the identical medium containing 10 μm MG-132, and the indicated concentration of 25-HC. Following incubation for 1 h at 37 °C, the cells were harvested, lysed in detergent-containing buffer, and immunoprecipitated with 30 μg of polyclonal IgG-839 (against reductase) as described under “Experimental procedures.” Aliquots of the resulting immunoprecipitates were then subjected to SDS-PAGE, followed by immunoblot analysis with IgG-A9 (against reductase) and IgG-P4D1 (against ubiquitin). The results in this and other figures are representative of at least two independent experiments. MW, molecular mass.

Figure 2.

Reduced synthesis of cholesterol in SV-589 cells lacking UBIAD1. SV-589 and SV-589(ΔUBIAD1) cells were set up on day 0 at a density of 4 × 10⁵ cells/60-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were switched to medium A containing 10% FCS. A, on day 2, the cells were refed the identical medium supplemented with 15 μCi/ml [¹⁴C]acetate; cold acetate was to obtain a final concentration of 0.5 mm. Following incubation for 4 h at 37 °C, the cells were harvested for preparation of lysates from which lipids were extracted and analyzed by TLC as described under “Experimental procedures.” Incorporation of [¹⁴C]acetate into [¹⁴C]cholesterol was determined by scintillation counting. A blank value, representing the amount of [¹⁴C]acetate incorporated into [¹⁴C]cholesterol in each cell line that was chilled to 4 °C, refed [¹⁴C]acetate-containing medium and immediately washed and extracted, was subtracted from each value. Each value is the mean of triplicate incubations (± standard error). B, on day 2, the cells were refed medium A supplemented with 10 μCi/ml [³H]mevalonolactone and either 10% FCS or LPDS. Following incubation for 4 h at 37 °C, the cells were harvested and lysed for lipid extraction; incorporation of [³H]mevalonolactone into [³H]cholesterol was determined as described in A. Each value is the mean of triplicate incubations (± standard error). The p values were calculated using Student's t test: NS, not significant; *, p ≤ 0.05; ***, p ≤ 0.001.

Figure 3.

Expression of SCD-associated UBIAD1 (N102S) leads to enhanced synthesis of cholesterol in SV-589 cells. SV589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at a density of 4 × 10⁵ cells/60-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were switched to medium A containing 10% FCS. On day 2, the cells received the identical medium supplemented with either 15 μCi/ml [¹⁴C]acetate (A) or 10 μCi/ml [³H]mevalonolactone (B) and incubated for 4 h at 37 °C. Incorporation of [¹⁴C]acetate and [³H]mevalonolactone into [³H]cholesterol was determined by TLC and scintillation counting as described in the legend to Fig. 2. Each value is the mean of triplicate incubations (± standard error). The p values were calculated using the Student's t test: NS, not significant; ***, p ≤ 0.001.

Figure 4.

Cholesterol ester synthesis and neutral lipid accumulation in SV-589 cells expressing wild-type and SCD-associated UBIAD1. A and B, SV589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 as described in the legend to Fig. 2. On day 1, the cells were refed medium A containing 10% DFCS. On day 2, the cells were refed the identical medium supplemented with 0.5 μCi/ml [³H]oleate and incubated for the indicated periods of time at 37 °C. Following incubations, the cells were harvested for preparation of lysates from which lipids were extracted and analyzed by TLC as described under “Experimental procedures.” Incorporation of [³H]oleate into [³H]cholesterol esters was determined by scintillation counting. The values were corrected for background as described in the legend to Fig. 2. Each value is the mean of triplicate incubations (± standard error). C, SV589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 3 × 10⁵ cells/60-mm dish with glass coverslips in medium A containing 5% FCS and 1 mm mevalonate. On day 1, the medium was switched to medium A supplemented with 10% FCS. On day 2, the cells were fixed and double-stained with 4′6-diamino-phenylindole for nuclei (blue) and with oil red O for neutral lipids (red) as described under “Experimental procedures.” D, quantification of the oil red O signal was performed using ImageJ. The values are the average of 10 images (± standard error). Scale bar, 20 μm. The p values were calculated using the Student's t test: ***, p ≤ 0.001.

Figure 5.

Mevalonate restores Golgi localization of UBIAD1 in compactin-treated SV-589 cells. SV-589 cells were set up on day 0 at 8 × 10⁴ cells/well of a 6-well dish containing glass coverslips in medium A supplemented with 5% FCS. On day 1, the cells were refed medium A supplemented with either 10% FCS or 10% LPDS, 10 μm compactin, and 0.05–10 mm mevalonate (A) or 0.3 μg/ml 25-HC (B) as indicated. Following incubation for ∼16 h at 37 °C, the cells were fixed and analyzed by immunofluorescence microscopy using IgG-1H12 (against human UBIAD1) and 4′6-diamino-phenylindole as described under “Experimental procedures.” Images were acquired as described in the legend to Fig. 4. Scale bars, 10 μm.

Figure 6.

The oxysterol 25-hydroxycholesterol enhances synthesis of MK-4. A, SV-589 cells were set up on day 0 at 2.5 × 10⁵ cells/60-mm dish in medium A supplemented with 10% FCS. On day 1, the cells were refed medium A supplemented with 10% LPDS, 10 μm compactin, 0.2 μCi/ml [³H]menadione, and the indicated concentration of mevalonate in the absence or presence of 0.3 μg/ml 25-HC. On day 2, the cells were harvested, the lipids were extracted, and the amount of [³H]menadione incorporated into MK-4 was determined by TLC and scintillation counting as described under “Experimental procedures.” The values are the means of triplicate samples (± standard error). B and C, SV-589 cells were set up on day 0 at 2.6 × 10⁴ cells/60-mm dish (for RNA isolation), 7 × 10⁵ cells/100-mm dish (subcellular fractionation) in medium A containing 10% FCS. On day 1, the cells were switched to medium A supplemented with 10% LPDS in the absence or presence of 10 μm compactin, 0.3 μg/ml 25-HC, and various concentrations of mevalonate as indicated. Following incubation for 16 h at 37 °C, the cells were harvested for isolation of total RNA (B) or subcellular fractionation (C) as described under “Experimental procedures.” B, total RNA from each condition was subjected to quantitative RT-PCR using primers against the indicated gene; 36B4 was used as an invariant control. Each value represents the amount of mRNA relative to that in untreated cells, which is arbitrarily defined as 1. The error bars represent ± standard error of triplicate samples. HMGCR, HMG CoA reductase; FPPS, farnesyl pyrophosphate synthase. C, resulting cytosolic, nuclear, and membrane fractions were subjected to SDS-PAGE (14–20 μg total protein/lane), followed by immunoblot analysis with antibodies against HMGCS, GGPPS, SREBP-2, nucleoporin, HMG CoA reductase, SQS, UBIAD1, actin, and calnexin.

Figure 7.

Flux through the mevalonate pathway in cells depleted of sterol and/or nonsterol isoprenoids. Shown is a schematic representation of flux through the mevalonate pathway when cells are depleted of both sterol and nonsterol isoprenoids (A) or replete with sterols and depleted of nonsterol isoprenoids (B) as determined by Faust et al. (18).