People with a rare genetic disorder called ‘Schnyder corneal dystrophy’ gradually lose their vision, because their corneas become increasingly cloudy. This cloudiness is caused by a build-up of excessive amounts of cholesterol, and the disorder itself is caused by mutations in a gene that encodes a protein called UBIAD1. Researchers have previously discovered that the UBIAD1 protein is involved in making vitamin K₂, but it is not clear how this protein also helps to control cholesterol levels in the cornea.

An enzyme called HMG CoA reductase makes a molecule that is used to make cholesterol and many other similar sterol molecules. A ‘feedback loop’ operates in cells to control the amount of the reductase and prevent cholesterol from becoming too high or too low. Sterol molecules, together with another molecule called geranylgeraniol, participate in this feedback loop by promoting the destruction of the reductase enzyme. Here, Schumacher et al. reveal a link between UBIAD1 and the reductase that may explain how UBIAD1 contributes to the production of excess cholesterol in patients with Schnyder corneal dystrophy.

The experiments show that, in the presence of sterol molecules, UBIAD1 can bind to HMG CoA reductase to protect the reductase from being destroyed by other proteins. Geranylgeraniol—which stops the UBIAD1 protein from binding to the enzyme—is required to completely destroy the reductase enzyme. However, when UBIAD1 is missing, the reductase enzyme is destroyed even in the absence of geranylgeraniol.

Furthermore, the experiments show that the genetic mutations linked to Schnyder corneal dystrophy lead to the production of versions of the UBIAD1 protein that bind to the reductase enzyme even when geranylgeraniol molecules are present. This prevents the normal breakdown of the reductase enzyme, which could lead to the build up of cholesterol in the cornea of individuals with the disorder.

Schumacher et al.'s findings show that the UBIAD1 protein helps to control the levels of cholesterol in cells by protecting the HMG CoA reductase enzyme from destruction. These findings may aid the development of new therapies to lower cholesterol levels in cells, which may help patients with Schnyder's corneal dystrophy and other conditions caused by high cholesterol levels.

SCD (Schnyder corneal dystrophy) is a rare autosomal dominant eye disease that is characterized by bilateral opacification of the cornea (Klintworth, 2009; Weiss, 2009). The clinical manifestation of SCD can be apparent early in life, usually within the first decade. Thereafter, opacification of the cornea progresses slowly and ultimately leads to reduced visual acuity, which is postulated to be caused by light scattering (Weiss, 2009). The significance of this visual impairment is highlighted by the frequency in which corneal transplant surgery is utilized in treatment of SCD; approximately 50% of SCD patients ≥50 years of age undergo corneal transplant surgery for restoration of normal vision acuity (Weiss, 2007). The frequency of the procedure increases to greater than 70% for SCD patients 70 years and older. Analyses of corneas removed from SCD patients during transplantation surgery revealed a marked accumulation of unesterified cholesterol and lesser accumulation of esterified cholesterol and phospholipids (McCarthy et al., 1994; Gaynor et al., 1996; Yamada et al., 1998), suggesting dysregulation of cholesterol metabolism may underlie pathogenesis of the disease. Systemic dyslipidemia has been reported to be associated with some, but not all cases of SCD (Thiel et al., 1977; Brownstein et al., 1991; Crispin, 2002).

In 2007, two groups independently identified SCD-associated mutations in the gene encoding UBIAD1 (UbiA prenyltransferase domain-containing protein-1) (Orr et al., 2007; Weiss et al., 2007). UBIAD1 (also known as TERE1) was first described as being absent or markedly diminished in bladder and prostate tumors (McGarvey et al., 2001, 2003) and belongs to the UbiA superfamily of prenyltransferases (Heide, 2009). These enzymes, which are found in a wide variety of species, contain 8–10 transmembrane helices and catalyze transfer of isoprenyl groups to aromatic acceptors, producing a diverse range of molecules including ubiquinone, chlorophylls, hemes, vitamin E, and vitamin K (Forsgren et al., 2004; Nakagawa et al., 2010; Bonitz et al., 2011). Indeed, UBIAD1 catalyzes transfer of the 20-carbon geranylgeranyl group from geranylgeranyl pyrophosphate to menadione (vitamin K₃) derived from plant-derived phylloquinone (vitamin K₁), generating MK-4 (menaquinone-4, vitamin K₂) (Figure 1) (Nakagawa et al., 2010; Hirota et al., 2013). It has also been proposed that UBIAD1 mediates polyprenylation of 4-hydroxybenzoate to produce 3-polyprenyl-4-hydroxybenzoate (PPHB), an intermediate in the synthesis of CoQ10 (coenzyme Q10 or ubiquinone-10) (Mugoni et al., 2013).

Figure 1

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To date, 24 UBIAD1 mutations have been identified in ∼50 SCD families (Nickerson et al., 2013; Nowinska et al., 2014). Several of these mutations alter amino acid residues that localize to the active site of the UBIAD1 prenyltransferase domain as determined by molecular modeling using structural models of bacterial and archaeal UbiA prenyltransferases (Bräuer et al., 2004, 2008; Nickerson et al., 2010; Cheng and Li, 2014; Huang et al., 2014). Cells from patients harboring four SCD-associated mutations (N102S, D112N, and G177E/R) exhibited reduced MK-4 biosynthetic activity (Nickerson et al., 2013). A possible link between UBIAD1 and cholesterol metabolism was provided by co-immunoprecipitation studies that showed an association of UBIAD1 with the cholesterol biosynthetic enzyme HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (Nickerson et al., 2013). However, the relevance of this association to regulation of cholesterol metabolism and pathogenesis of SCD is not clear.

In mammalian cells, the ER (endoplasmic reticulum)-localized reductase catalyzes reduction of HMG CoA to mevalonate (Figure 1). This reaction constitutes a rate-limiting step in synthesis of cholesterol as well as nonsterol isoprenoids such as the farnesyl and geranylgeranyl groups that are transferred to many cellular proteins and utilized in synthesis of ubiquinone, heme, and dolichol (Goldstein and Brown, 1990; Casey and Seabra, 1996). Multiple feedback mechanisms converge on reductase to ensure a constant supply of nonsterol isoprenoids, while avoiding overaccumulation of cholesterol (Brown and Goldstein, 1980). This feedback system is exploited therapeutically by a group of competitive inhibitors of reductase called statins, which trigger regulatory responses that result in the lowering of plasma levels of LDL (low-density lipoprotein)-cholesterol and thereby reduce the incidence of cardiovascular disease (Stossel, 2008; Marais et al., 2014). Statins also block production of sterol and nonsterol isoprenoids that govern feedback regulation of reductase (Goldstein and Brown, 1990). Thus, a marked accumulation of reductase occurs in livers of statin-treated animals and humans (Brown and Goldstein, 1980; Kita et al., 1980; Reihner et al., 1990), which can blunt the cholesterol-lowering effects of these drugs. This accumulation results in part, from slowed ERAD (ER-associated degradation) of reductase (Nakanishi et al., 1988; Inoue et al., 1991; Ravid et al., 2000).

The ERAD of reductase is initiated by intracellular accumulation of sterols, which causes the enzyme to bind to ER membrane proteins called Insig-1 and Insig-2 (Sever et al., 2003a, 2003b) (Figure 2). Insig binding occurs through the membrane domain of reductase, which contains eight membrane-spanning helices and precedes a large cytosolic domain with enzymatic activity (Liscum et al., 1985; Roitelman et al., 1992). Insig-associated ubiquitin ligases gp78 and Trc8 facilitate ubiquitination of cytosolically exposed lysine residues in the membrane domain of reductase (Sever et al., 2003a; Song et al., 2005; Jo et al., 2011; Liu et al., 2012). This ubiquitination marks reductase for recognition by the AAA (ATPases associated with diverse cellular activities)-ATPase VCP/p97, which mediates extraction of reductase across ER membranes (Morris et al., 2014). Once extracted, ubiquitinated reductase is then released into the cytosol and delivered into the proteolytic chamber of the 20S proteasome through reactions mediated by the proteasome 19S regulatory particle, which contains six AAA-ATPases (Ehlinger and Walters, 2013). Geranylgeraniol, the alcohol derivative of geranylgeranyl pyrophosphate, augments sterol-accelerated ERAD of reductase but does not appreciably affect sterol-induced ubiquitination (Sever et al., 2003a). This observation led to the notion that geranylgeraniol augments post-ubiquitination steps in reductase ERAD (Figure 2). Indeed, geranylgeraniol enhances sterol-induced membrane extraction and cytosolic dislocation of reductase as judged by assays carried out in vitro and in intact cells (Song and DeBose-Boyd, 2004; Elsabrouty et al., 2013; Morris et al., 2014).

Figure 2

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In the current studies, we use proximity-dependent biotinylation (Roux et al., 2012) to identify UBIAD1 as an associated protein of the membrane domain of reductase, which is necessary and sufficient for sterol-accelerated ERAD (Gil et al., 1985; Skalnik et al., 1988). Endogenous UBIAD1 binds to reductase in sterol-treated cells as determined by co-immunoprecipitation. This sterol-induced binding is inhibited by geranylgeraniol, releasing UBIAD1 for translocation from membranes of the ER to the Golgi. CRISPR/Cas9-mediated knockout and RNA interference (RNAi)-mediated knockdown of UBIAD1 relieves the geranylgeraniol requirement for reductase ERAD. UBIAD1 harboring the SCD-associated N102S mutation resists geranylgeraniol-mediated displacement from reductase and remains associated with the protein in the ER, thereby blocking sterol-accelerated degradation of reductase. These studies identify UBIAD1 as the elusive target of geranylgeraniol in the ERAD of reductase and provide insight into mechanisms through which the nonsterol isoprenoid modulates the reaction. Moreover, our results suggest that slowed ERAD of reductase may contribute to the accumulation of cholesterol observed in SCD.

To identify proteins that associate with reductase, we employed a novel chimeric protein composed of the membrane domain of reductase (amino acids 1–346) fused to the promiscuous biotin ligase BirA*, which features proximity-dependent biotinylation of the chimera's near-neighbor proteins (Roux et al., 2012). Cells stably expressing high levels of the reductase-BirA* chimera were subjected to treatment with the oxysterol 25-HC (25-hydroxycholesterol) in the presence of biotin. Following treatments, cells were harvested and lysed; the lysates were then incubated with streptavidin-conjugated agarose beads to capture biotinylated proteins that were subsequently identified by tandem mass spectrometry.

Figure 3—figure supplement 1B shows an experiment in which we used co-immunoprecipitation to measure the interaction of endogenous reductase with several proteins that were identified as associated with the reductase-BirA* chimera (Figure 3—figure supplement 1A). SV-589 cells, a line of transformed human fibroblasts (Yamamoto et al., 1984), were depleted of sterols and subsequently treated in the absence or presence of 25-HC. Following treatments, the cells were harvested for lysis in detergent-containing buffer; the resulting lysates were then immunoprecipitated with polyclonal antibodies against reductase. Immunoblot analysis revealed that treatment of cells with 25-HC triggered co-immunoprecipitation of endogenous reductase with Insig-1 (Figure 3—figure supplement 1B, lanes 3 and 4); the control ER membrane protein calnexin failed to interact with reductase, regardless of the absence or presence of 25-HC (lanes 5 and 6). ERGIC-53 similarly failed to appear in the anti-reductase immunoprecipitates (lanes 9 and 10). In contrast, lamina-associated peptide-2 (lanes 7 and 8), peroxiredoxin-4 (lanes 11 and 12), PGRMC2 (lanes 13 and 14), and annexin-A1 (lanes 15 and 16) co-precipitated with reductase in both the absence and presence of 25-HC; the significance and specificity of these interactions are unclear. In the absence of 25-HC, a small amount of UBIAD1 (UbiA prenyltransferase domain-containing protein-1) appeared in the pellet fraction of the reductase immunoprecipitation (lane 17); this appearance was markedly enhanced when the cells were subjected to treatment with 25-HC (lane 18).

UBIAD1 belongs to the UbiA superfamily of prenyltransferases (Heide, 2009; Bonitz et al., 2011). Similar to other UbiA prenyltransferases, UBIAD1 is very hydrophobic; the human enzyme is comprised of 338 amino acids and is predicted to contain at least eight transmembrane helices (Figure 3A). Besides its regulated association with reductase (this study and Nickerson et al., 2013), we chose UBIAD1 for further examination owing to the observation that mutations in the UBIAD1 gene are associated with SCD, which may result from dysregulated cholesterol metabolism (McCarthy et al., 1994; Gaynor et al., 1996; Yamada et al., 1998). The experiment of Figure 3B shows a time course of 25-HC-mediated association of UBIAD1 with reductase as determined by co-immunoprecipitation. Immunoblot analysis of anti-reductase immunoprecipitates revealed co-precipitation of endogenous UBIAD1 with reductase following treatment of cells for 30 min with 25-HC (Figure 3B, top panel, lane 6). This sterol-induced co-precipitation continued after prolonged incubation (top panel, lanes 7–14), even though the total amount of reductase was reduced by accelerated degradation (second and fourth panels, lanes 12 and 14). The structurally related 1,1-bisphosphonate esters SR-12813 and Apomine mimic 25-HC in accelerating reductase ubiquitination and degradation (Roitelman et al., 2004; Sever et al., 2004; Nguyen et al., 2009). As shown in Figure 3C, Apomine triggered co-precipitation between reductase and UBIAD1 in a manner similar to that of 25-HC (second panel, compare lane 2 with lanes 3–5).

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We next used RNA interference (RNAi) to examine a role for Insigs in the 25-HC-induced binding of reductase to UBIAD1. SV-589 cells were transfected with duplexes of small interfering RNAs (siRNAs) against mRNAs encoding green fluorescent protein (GFP), which is not expressed in the cells, or Insig-1 and Insig-2. Following transfection, the cells were depleted of sterols, incubated in the absence or presence of 25-HC, and lysed for subsequent immunoprecipitation with polyclonal anti-reductase. Immunoblotting of precipitated proteins revealed that 25-HC stimulated co-precipitation of both UBIAD1 and Insig-1 with reductase in SV-589 cells transfected with control GFP siRNA (Figure 4A, second and third panels, lane 2). The sterol-induced co-precipitation of UBIAD1 with reductase was markedly reduced in cells that received siRNAs against Insig-1 and Insig-2 (second panel, lanes 3 and 4). The experiment of Figure 4B shows that knockdown of UBIAD1 reduced, but did not eliminate the sterol-induced binding of reductase to Insig-1 (third panel, compare lanes 2 and 4). Finally, we examined the requirement of reductase for the association of UBIAD1 with Insig-1. Immunoblot analysis of anti-UBIAD1 immunoprecipitates revealed that as expected, reductase and Insig-1 co-precipitated with UBIAD1 when control siRNA-transfected cells were treated with 25-HC (Figure 4C, top and bottom panels, lane 2). However, RNAi-mediated knockdown of reductase abolished co-precipitation of Insig-1 with UBIAD1 (bottom panel, compare lane 4 with lane 2).

Figure 4

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Results of Figure 3B show that UBIAD1 continued to appear in anti-reductase immunoprecipitates after prolonged treatment with 25-HC, even though total reductase was reduced by accelerated degradation. We reasoned that the proteasome inhibitor MG-132 would block this degradation, leading to increased co-immunoprecipitation of UBIAD1 with stabilized reductase. Immunoblot analysis of anti-reductase immunoprecipitates from lysates of cells treated in the absence of MG-132 shows that 25-HC stimulated reductase degradation (Figure 5—figure supplement 1A, top panel, compare lanes 1 and 2), which was blocked by MG-132 (lanes 3 and 4). Insig-1 co-precipitated with reductase in the presence of 25-HC (second panel, lane 2) and this co-precipitation was enhanced by MG-132 (lane 4). UBIAD1 also co-precipitated with reductase in 25-HC-treated cells (third panel, lane 2); however, the interaction was reduced, rather than enhanced, in the presence of MG-132 (lane 4).

Taking into consideration results of Figure 5—figure supplement 1A together with the previous observation that geranylgeraniol augments post-ubiquitination steps in reductase ERAD (Sever et al., 2003a; Elsabrouty et al., 2013), we next sought to determine whether geranylgeraniol modulates UBIAD1-reductase binding. As expected, 25-HC dose-dependently stimulated co-precipitation of UBIAD1 with reductase (Figure 5A, top panel, lanes a–c); however, this sterol-regulated association was inhibited in the presence of 10 or 20 µM geranylgeraniol (lanes d–i). Geranylgeraniol-mediated inhibition of reductase-UBIAD1 binding was also observed in the repeat experiment shown in Figure 5—figure supplement 1B. In contrast, sterol-induced binding of UBIAD1 to reductase continued in the presence of 20 µM farnesol (Figure 5B, top panel, compare lanes a–d with lanes e–h), a 15-carbon nonsterol isoprenoid that does not augment sterol-accelerated ERAD of reductase (Sever et al., 2003a).

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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 role of UBIAD1 in sterol-accelerated reductase ERAD was next examined using RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout (Cong et al., 2013; Mali et al., 2013). For RNAi experiments, SV-589 cells were transfected with siRNA duplexes targeting the GFP or UBIAD1 mRNAs and subsequently treated in the absence or presence of 25-HC and geranylgeraniol prior to subcellular fractionation. Figure 6A shows that 25-HC stimulated reductase degradation from membranes of control siRNA-transfected cells (top panel, lane 2); this degradation was enhanced by geranylgeraniol (lanes 3 and 4). In UBIAD1 knockdown cells, 25-HC stimulated complete degradation of reductase, even in the absence of geranylgeraniol (lanes 5–8). Nearly identical results were obtained using an siRNA duplex targeting a different region of the UBIAD1 mRNA (Figure 6—figure supplement 1). Geranylgeraniol also augmented the ERAD of reductase that was stimulated by Apomine (Figure 6B, top panel, lanes 1–4); however, the 1,1-bisphosphonate ester caused reductase to become completely degraded in UBIAD1 knockdown cells (lanes 5–8). Results consistent with those observed using RNAi were obtained in cells subjected to CRISPR/Cas9-mediated knockout of UBIAD1. In wild type SV-589 cells, Apomine stimulated reductase ERAD through a mechanism augmented by geranylgeraniol (Figure 6C, top panel, lanes 1–4), whereas the compound caused complete degradation of reductase in cells deficient in UBIAD1 (designated UBIAD1⁻) (lanes 5–8). Figure 6D shows that in a time-dependent fashion, geranylgeraniol augmented Apomine-induced degradation of reductase in SV-589 cells (compare lanes 1–6 with lanes 7–12). However, Apomine alone caused complete degradation of reductase in UBIAD1⁻ cells (Figure 6D, lanes 13–18) and augmentation by geranylgeraniol was markedly diminished (lanes 19–24). Importantly, transfection of UBIAD1⁻ cells with pCMV-Myc-UBIAD1 restored the requirement of geranylgeraniol for maximal degradation of reductase (Figure 6E, top panel, compare lanes 1–3 with lanes 4–6).

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To date, 24 mutations in the UBIAD1 gene have been identified in SCD families (Nickerson et al., 2013; Nowinska et al., 2014); these mutations result in the alteration of 20 amino acid residues in UBAID1 (Figure 3A). Asparagine-102 (N102), which localizes to the prenyltransferase domain of UBIAD1 (Cheng and Li, 2014; Huang et al., 2014), is the most frequently altered residue (∼30%) in SCD families. Considering that SCD is an autosomal dominant disorder, we reasoned that overexpression of UBIAD1 harboring the SCD-associated N102S mutation would block reductase ERAD. Figure 7A shows that when overexpressed in SV-589 cells, the T7-tagged membrane domain of reductase became degraded in the presence of 25-HC and geranylgeraniol (top panel, lanes 1 and 2). Co-expression of a plasmid encoding wild type UBIAD1 stabilized reductase in a dose-dependent fashion and 25-HC plus geranylgeraniol continued to accelerate its ERAD (lanes 3–10). Notably, the highest level of wild type UBIAD1 co-expressed with reductase blunted the enzyme's accelerated degradation (lanes 11 and 12). Co-expression of UBIAD1 (N102S) also stabilized the membrane domain of reductase (Figure 7B, top panel, compare lane 1 with lanes 3, 5, 7, 9, and 11), suggesting the protein continued to bind to the enzyme. However, UBIAD1 (N102S) blunted sterol-accelerated ERAD of reductase, even at low levels of expression (Figure 7B, top panel, compare lane 2 with lanes 4, 8, 10, and 12). Similar results were obtained with T7-tagged, full-length reductase (Figure 7—figure supplement 1). The protein was subjected to sterol-accelerated degradation when transfected alone or together with up to 100 ng of plasmid encoding wild type UBIAD1 (Figure 7—figure supplement 1A, top panel, lanes 1–8); co-transfection of higher levels (300 and 1000 ng) of the UBIAD1-encoding plasmid inhibited reductase degradation (lanes 9–12). Consistent with results obtained with the reductase membrane domain, inhibition of full-length reductase degradation was observed upon co-transfection with a significantly lower amount (30 ng) of plasmid encoding UBIAD1 (N102S) (Figure 7—figure supplement 1B, top panel, compare lanes 1–4 with lanes 5–12). Glycine-177 (G177, see Figure 3A) is the second-most frequently altered UBIAD1 residue in SCD families (Nickerson et al., 2013). The results of Figure 7C show that UBAID1 harboring the SCD-associated G177R mutation blocked reductase ERAD to a degree similar to that observed with UBIAD1 (N102S) (top panel, compare lanes 1 and 2 with lanes 3–6).

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We next compared the geranylgeraniol-mediated displacement of wild type and N102S UBIAD1 from reductase using co-immunoprecipitation. UBIAD1⁻ cells stably transfected with empty mock vector, pCMV-Myc-UBIAD1, or pCMV-Myc-UBIAD1 (N102S) were briefly incubated with 25-HC in the absence or presence of geranylgeraniol. The cells were then harvested, lysed, and subjected to anti-reductase immunoprecipitation. The results show that wild type UBIAD1 co-precipitated with reductase in the 25-HC-treated cells (Figure 8A, second panel, lane e). This co-precipitation was inhibited by geranylgeraniol (lanes g and h). UBIAD1 (N102S) similarly co-precipitated with reductase (lane i); however, the mutant UBAID1 resisted geranylgeraniol-mediated displacement and remained associated with reductase (lanes j–l). UBIAD1 (N102S) exhibited a similar resistance to geranylgeraniol-mediated displacement from reductase in two independent experiments shown in Figure 8—figure supplement 1.

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In the experiment of Figure 8B, we used immunofluorescence to examine the subcellular localization of wild type and SCD-associated mutants of UBIAD1 in SV-589 cells grown in FCS-containing medium. The results show that wild type UBIAD1 localized to Golgi membranes that also stained with antibodies against GM130 (Figure 8B, panels 1–4). UBIAD1 (N102S) primarily exhibited a diffuse, reticular localization corresponding to ER membranes and little, if any, of the protein appeared in the Golgi (Figure 8B, panels 5–8). The repeat experiment shown in Figure 8—figure supplement 2 confirms that wild type UBIAD1 localized to the Golgi, whereas UBIAD1 (N102) primarily localized to ER membranes. Similar to the N102 mutant, UBIAD1 (G177R) was also primarily concentrated in membranes of the ER, but not the Golgi (Figure 8B, panels 9–12).

Results of the current experiments form the basis for the model shown in Figure 9 that depicts the role of UBIAD1 in sterol-accelerated ERAD of reductase. As previously proposed (Morris et al., 2014), the reaction is initiated by accumulation of sterols, which triggers binding of Insigs to reductase and results in its gp78/Trc8-mediated ubiquitination (Song et al., 2005; Jo et al., 2011). We find in the current study that the oxysterol 25-HC and the 1,1-bisphosphonate ester Apomine, which mimics 25-HC in accelerating reductase ERAD (Roitelman et al., 2004; Sever et al., 2004; Nguyen et al., 2009), trigger binding of reductase to UBIAD1 (Figure 3B,C), a prenyltransferase that mediates synthesis of the vitamin K₂ derivative MK-4 (see Figure 1). The sterol-induced binding of UBIAD1 to reductase appears to follow the action of Insigs as indicated by inhibition of the association by RNAi-mediated Insig knockdown (Figure 4A). In contrast to the action of sterols, the nonsterol isoprenoid geranylgeraniol, which augments sterol-accelerated reductase ERAD (Sever et al., 2003a), inhibits formation of the UBIAD1-reductase complex (Figure 5A and Figure 5—figure supplement 1B). Geranylgeraniol also regulates the subcellular localization of UBIAD1. The prenyltransferase primarily localizes to Golgi membranes when cells are cultured in sterol-replete FCS-containing medium (Figure 5C). The Golgi localization of UBIAD1 is disrupted when cells are switched to sterol-depleting medium containing LPPS and the reductase inhibitor compactin, which depletes cellular stores of nonsterol isoprenoids (Brown and Goldstein, 1980). Remarkably, Golgi localization of UBAID1 is restored by the addition of geranylgeraniol, but not of 25-HC or farnesol, to cells deprived of sterol and nonsterol isoprenoids (Figure 5C and Figure 5—figure supplements 2, 3). We conclude from these observations that geranylgeraniol-mediated disruption of the UBIAD1-reductase complex allows for the translocation of UBIAD1 from ER membranes to the Golgi and for membrane extraction, cytosolic dislocation and proteasomal degradation of reductase (Figure 9).

Figure 9

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The model shown in Figure 9 predicts that UBIAD1 inhibits sterol-accelerated ERAD of reductase and that geranylgeraniol relieves this inhibition by triggering displacement of UBIAD1 from the protein. In support of this notion, we observed that RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of UBIAD1 eliminates the requirement of geranylgeraniol for maximal degradation of reductase (Figure 6). The continuance of accelerated reductase degradation in UBIAD1-deficient cells is consistent with the proposal that UBIAD1 mediates a step in the reaction downstream the action of Insigs. In future studies, efforts will be undertaken to delineate mechanisms through which UBIAD1 retards reductase ERAD. Geranylgeraniol is required for extraction of ubiquitinated reductase across the ER membrane through a reaction that is mediated by the AAA-ATPase VCP/p97 (Morris et al., 2014). Thus, one likely mechanism for the inhibitory effects of UBIAD1 on reductase ERAD may involve inhibition of the association of ubiquitinated reductase with VCP/p97 or one of its ubiquitin-binding cofactors, thereby preventing its subsequent membrane extraction.

In addition to revealing the molecular mechanism through which geranylgeraniol modulates reductase ERAD, the current results help to explain how cells maintain synthesis of nonsterol isoprenoids, while avoiding overproduction of cholesterol. In 1979, Faust, Brown, and Goldstein found that in the absence of LDL, cells accumulate reductase to produce mevalonate for conversion into primarily cholesterol and other sterols (Faust et al., 1979). The addition of LDL to cells partially suppressed reductase, limiting production of mevalonate. As a result, incorporation of mevalonate into cholesterol was reduced, whereas incorporation of the molecule into nonsterol isoprenoids such as CoQ10 was enhanced. We postulate that LDL failed to completely suppress reductase due to its binding to UBIAD1 and resultant inhibition of sterol-accelerated degradation. This residual reductase produces a small amount of mevalonate that is preferentially diverted into nonsterol isoprenoids. Once appropriate levels of geranylgeranyl pyrophosphate accumulate in cells, the isoprenoid binds to UBIAD1, causing its release from reductase, which subsequently becomes degraded. Released UBIAD1 then translocates to the Golgi where it synthesizes PPHB (Mugoni et al., 2013) and perhaps MK-4. Thus, substrate-regulated transport of UBIAD1 ensures that products of the enzyme are only produced when cellular demands for other nonsterol isoprenoids as well as sterols have been met. Studies are currently underway to appraise this notion and to determine whether Golgi-localized UBIAD1 is enzymatically active to synthesize MK-4 and PPHB, whereas the ER-localized enzyme is not. Moreover, the role of reductase in ER to Golgi trafficking of UBAD1 will be investigated.

The significance of geranylgeraniol-mediated displacement of UBIAD1 from reductase is revealed through the study of SCD-associated mutants of the prenyltransferase. Consistent with the autosomal dominant phenotype of SCD, disease-associated N102S and G177R mutants of UBIAD1 inhibit sterol-accelerated ERAD of reductase (Figure 7B,C, and Figure 7—figure supplement 1). The molecular basis for this inhibition is suggested by Figure 8A and Figure 8—figure supplement 1, which show that UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from reductase. Subcellular localization studies show that in FCS-cultured cells, wild type UBIAD1 localizes to the Golgi, whereas the N102S and G177R mutants of the enzyme appear defective in Golgi trafficking and remain sequestered within the ER (Figure 8B). Mutation of the asparagine residue in bacterial UbiA prenyltransferases that corresponds to N102 markedly diminishes substrate binding and enzymatic activity (Cheng and Li, 2014; Huang et al., 2014). Thus, we postulate that binding of UBIAD1 to geranylgeranyl pyrophosphate produced by phosphorylation of geranylgeraniol triggers displacement of wild type UBIAD1 from reductase, allowing the protein to translocate to the Golgi. In contrast, the N102S and G177R mutants of UBIAD1 exhibit reduced affinity for geranylgeranyl pyrophosphate and thus, remain associated with reductase in the ER, thereby inhibiting its sterol-accelerated ERAD (Figure 9). This inhibition may contribute to the accumulation of cholesterol that is observed in corneas of SCD patients. It will be important in future studies to develop a geranylgeranyl pyrophosphate-binding assay for mammalian UBIAD1 and to determine the subcellular localization and effect of the other 18 SCD-associated UBIAD1 mutants on reductase ERAD.

In conclusion, the current study has identified UBIAD1 as the elusive molecular target of geranylgeraniol in reductase ERAD. They have also revealed a potential mechanism whereby mutations in UBIAD1 cause the accumulation of cholesterol in corneas of SCD patients. Finally, results of this study indicate the existence of a novel link between the synthesis of MK-4 and cholesterol. Further investigation of this link is merited owing to the potential for development of novel therapies that accelerate reductase ERAD to lower plasma LDL-cholesterol and retard corneal accumulation of cholesterol that characterizes SCD.

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Author details

1. Marc M Schumacher

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

   Contribution

   MMS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Rania Elsabrouty

   Competing interests

   The authors declare that no competing interests exist.

2. Rania Elsabrouty

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

   Contribution

   RE, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Marc M Schumacher

   Competing interests

   The authors declare that no competing interests exist.

3. Joachim Seemann

   Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   JS, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Youngah Jo

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

   Contribution

   YJ, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Russell A DeBose-Boyd

   Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   RADB, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Russell.Debose-Boyd@utsouthwestern.edu

   Competing interests

   The authors declare that no competing interests exist.

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