Schnyder corneal dystrophy-associated UBIAD1 mutations cause corneal cholesterol accumulation by stabilizing HMG-CoA reductase

Schnyder corneal dystrophy (SCD) is a rare genetic eye disease characterized by corneal opacification resulted from deposition of excess free cholesterol. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is an enzyme catalyzing biosynthesis of coenzyme Q10 and vitamin K2. More than 20 UBIAD1 mutations have been found to associate with human SCD. How these mutants contribute to SCD development is not fully understood. Here, we identified HMGCR as a binding partner of UBIAD1 using mass spectrometry. In contrast to the Golgi localization of wild-type UBIAD1, SCD-associated mutants mainly resided in the endoplasmic reticulum (ER) and competed with Insig-1 for HMGCR binding, thereby preventing HMGCR from degradation and increasing cholesterol biosynthesis. The heterozygous Ubiad1 G184R knock-in (Ubiad1G184R/+) mice expressed elevated levels of HMGCR protein in various tissues. The aged Ubiad1G184R/+ mice exhibited corneal opacification and free cholesterol accumulation, phenocopying clinical manifestations of SCD patients. In summary, these results demonstrate that SCD-associated mutations of UBIAD1 impair its ER-to-Golgi transportation and enhance its interaction with HMGCR. The stabilization of HMGCR by UBIAD1 increases cholesterol biosynthesis and eventually causes cholesterol accumulation in the cornea.

The UBIAD1 protein belongs to the UbiA superfamily of intramembrane aromatic prenyltransferases, which catalyze the biosynthesis of a variety of lipophilic molecules such as ubiquinones, vitamin K, vitamin E, chlorophylls, hemes and archaeal tetraether lipids [10]. UBIAD1 has been identified as a vitamin K 2 biosynthesis enzyme in humans and mice, and is essential for mouse embryonic development [11,12]. In Drosophila, the UBIAD1 homolog was reported to generate vitamin K 2 that functions as an electron carrier for sustaining mitochondrial function [13]. In zebrafish, UBIAD1 was proposed to synthesize ubiquinone coenzyme Q10 that protects against oxidative damages through regulating nitric oxide activity, thereby maintaining vascular endothelial cell survival [14]. However, how UBIAD1 mutations cause cholesterol accumulation in the cornea is not fully understood.
The endoplasmic reticulum (ER)-localized 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMG-CoA reductase, HMGCR) is a rate-limiting enzyme of the cholesterol biosynthetic pathway catalyzing the synthesis of mevalonate [15]. As an important intermediate, mevalonate gives rise to not only cholesterol, but also nonsterol isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP) that further generates ubiquinone, dolichol and hemes [15]. Interestingly, these mevalonate-derived molecules are also the final products of UBIAD1 superfamily members, suggesting a potential link between HMGCR and UBIAD1. Theses sterols and nonsterol isoprenoids coordinate to accelerate degradation of HMGCR to prevent over-accumulation of cholesterol [16]. Accumulating sterols induce HMGCR binding to ER-anchored Insig-1 and Insig-2, which bring ubiquitin ligases gp78, TRC8 and RNF145 together with other cofactors to ubiquitinate HMGCR, resulting in its degradation in proteasome [17][18][19][20][21][22]. Elucidating the molecular pathway of HMGCR degradation is of potential clinic significance. Statins as competitive inhibitors of HMGCR can decrease the synthesis of sterols and nonsterol isoprenoids and dramatically increase HMGCR in the liver [23,24]. The lanosterol analog HMG499 (also named Cmpd 81) can potentiate the cholesterol-lowering effect of statins through inducing HMGCR degradation [25].
Recently, Ubiad1 was found to be a HMGCR-binding protein through proximity-dependent biotinylation using HMGCR as the bait [26]. The homozygous Ubiad1 N100S knock-in (Ubiad1 N100S/N100S ) mice exhibit corneal opacification and HMGCR accumulation in tissues [9]. In this study, we identified HMGCR as a UBIAD1-associated protein through UBIAD1 immunoprecipitation coupled with mass spectrometry. We demonstrated that UBIAD1 competed with Insig-1 for binding HMGCR, preventing the latter from ubiquitination and degradation. All known SCD-associated UBIAD1 mutants localized in the ER and stabilized HMGCR protein. More importantly, we generated a different SCD-associated knock-in mouse line carrying Ubiad1 G184R mutation. The Ubiad1 G184R/+ mice exhibited excess HMGCR protein in tissues and striking corneal opacifications with free cholesterol deposition. These phenotypes recapitulate clinical manifestations of human SCD patients, suggesting that Ubiad1 G184R/+ mouse is an ideal model to study human SCD.

SCD-associated G186R UBIAD1 blocks degradation of HMGCR
To explore the underlying connections between UBIAD1 and cholesterol metabolism, we performed a tandem affinity purification (TAP) coupled to mass spectrometry to identify UBIA-D1-associated proteins, using HEK-293 cells stably expressing human wild-type (WT) or G186R mutant form of UBIAD1 fused with a TAP tag at the C terminus (S1A Fig). The G186R mutation is a mutation found in an early-onset SCD family [27]. Besides, according to the determined structure of archaeal UbiA, the G186 residue was proposed to locate in the surface-exposed loop, and the G186R mutation may affect the interactions with other proteins [10,28]. We found three proteins among the top of the list: HMGCR, VCP/p97 and SEL1L (S1B and S1C Fig, S1 Table). HMGCR is the rate-limiting enzyme converting HMG-CoA to mevalonate in the cholesterol biosynthetic pathway [15]. VCP/p97 and SEL1L have been known to be involved in the degradation of HMGCR and other ER proteins [29][30][31].
We next analyzed the effect of Ubiad1 on HMGCR ubiquitination. 25-HC triggered pronounced ubiquitination of immunoprecipitated HMGCR in the presence of proteasome inhibitor MG-132, which, however, was markedly reduced by WT Ubiad1 (Fig 1C, second panel, compare lane 2 and 4). The Ubiad1 (G184R) further inhibited the ubiquitination of HMGCR ( Fig 1C, second panel, compare lane 4 and 6). The total amount of HMGCR from input was more abundant in Ubiad1 (G184R)-expressing cells than those expressing Ubiad1 (WT) ( Fig  1C, third panel, lanes 3-6), consistent with results in Fig 1A and 1B.
We then used co-immunoprecipitation experiments to address potential interaction between HMGCR, Insig-1 and Ubiad1. The results showed that HMGCR pulled down more amounts of Ubiad1 (G184R) than Ubiad1 (WT) (Fig 1D). Meanwhile, Insig-1 association with Effects of human wild-type (WT) UBIAD1 and mutated UBIAD1 (G186R) (equivalent to mouse G184R) on HMGCR protein level. HEK-293 cells stably expressing human (homo sapiens, Hs) WT UBIAD1-TAP and UBIAD1 (G186R)-TAP, respectively, were depleted of sterols in sterol-depleted medium containing 10% LPDS, 1 μM lovastatin and 50 μM mevalonate for 16 hr. Then indicated concentrations of 25-hydroxylcholesterol (25-HC) were added and incubated for 5 hr. Cells were harvested and subjected to immunoblot with antibodies against HMGCR (A9), SREBP-2 (1D2), and UBIAD1 (anti-Flag). Clathrin heavy chain (CHC) was a loading control. TAP: Flag-Protein A tags for tandem affinity purification in S1 Fig. (B) Effects of mouse WT and G184R Ubiad1 on the degradation of the ubiquitin sites mutated (K89R/K248R) HMGCR. CHO-K1 cells were transfected with indicated plasmids, depleted of sterols for 16 hr and treated with 1 μg/ml 25-HC and 10 mM mevalonate (Mev) for 5 hr. Lysates were immunoblotted with indicated antibodies. Mus musculus, Mm. (C) Effects of WT and G184R Ubiad1 on the ubiquitination of HMGCR. CHO-K1 cells were transfected and depleted of sterols, then cells were treated with1 μg/ml 25-HC, 10 mM mevalonate and 20 μM MG-132 for 2 hr before immunoprecipitation. Lysates were immunoprecipitated with anti-T7 antibody coupled agarose, and pellets and inputs were probed for indicated proteins. (D) Interaction between Ubiad1 and HMGCR. CHO-K1 cells were transfected, depleted of sterols and treated with 25-HC for 1 hr, then lysates were immunoprecipitated with anti-T7 antibody coupled agarose. Pellets and inputs were probed with indicated antibodies. The experiments are repeated three times and representative data are shown.
HMGCR was reduced ( Fig 1D). Together, these results suggest that SCD-associated mutant Ubiad1 competes with Insigs to bind HMGCR, thereby blocking Insig-mediated ubiquitination and degradation of HMGCR.

UBIAD1 (G186R) enhances cholesterol synthesis
We then analyzed whether SCD-associated UBIAD1 (G186R) had any effect on cellular cholesterol level. Using the colorimetric method, we measured the amount of total cholesterol in cells stably expressing WT or G186R form of UBIAD1. UBIAD1 (G186R)-expressing cells indeed had more cholesterol than control (Fig 2A). However, no difference in the amount of nonesterified fatty acid (NEFA) was detected between these two cell lines (Fig 2B). We next sought to determine whether the G186R mutation increased de novo synthesis of cholesterol by using [ 14 C]-acetate to label newly synthesized cholesterol and fatty acid. [ 14 C]-cholesterol was markedly increased in UBIAD1 (G186R)-expressing cells (Fig 2C), whereas [ 14 C]-fatty acid remained comparable between WT-and UBIAD1 (G186R)-expressing cells (Fig 2D). After depletion, cells were washed with PBS to remove lovastatin, and change to medium with 10% delipidated-FCS for 5 hr. Then the lipids were extracted and total cholesterol (A) and non-esterified fatty acids (NEFA) (B) were measured. The protein levels were quantified by BCA method, and used to normalize the lipid content. The data are shown as mean ± SD, n = 3. The p value was calculated with Student's t test; � , p < 0.05. (C-D) Effects of WT and UBIAD1 (G186R) on de novo synthesis of cholesterol (C) and fatty acid (D). HEK-293 cells stably expressing with WT and G186R UBIAD1, were depleted of sterols and fatty acids in 10% delipidated-FCS, 1 μM lovastatin and 50 μM mevalonate for 16 hr. After depletion, cells were washed to remove lovastatin, and change to medium with 10% delipidated-FCS for 3 hr. Then [ 14 C]-acetate (36 μCi/100-mm dish) were added and cells were treated for additional 2 hr. Cholesterol (C) and fatty acid (D) were extracted and resolved by thin-layer chromatography. Radioactive signals were visualized with phosphoimager. Signal intensities were quantified by Image-Pro Plus 6 software, and the signals in WT UBIAD1 expressing cells were defined as 1. All experiments are repeated three times and representative data are shown. These results together suggest that the UBIAD1 (G186R) mutation enhances synthesis of cholesterol.

All SCD-associated UBIAD1 mutants reduce degradation of HMGCR
Human UBIAD1 contains 338 amino acids with 8 predicated transmembrane helices, and 21 SCD-associated UBIAD1 nucleotide mutations in the coding sequence that altered amino acids at 19 positions are shown in Fig 3A. Protein sequence analysis showed that amino acids that are mutated in SCD are evolutionarily conserved from fly to human (S2 Fig). As UBIAD1 (G186R) dramatically stabilized HMGCR and increased cellular cholesterol level (Fig 1, Fig 2), we next examined the effect of other SCD-associated UBIAD1 mutations on sterol-induced degradation of HMGCR. Each of the 21 SCD-associated missense mutations of UBIAD1 were co-transfected with HMGCR and Insig-1 expression plasmids individually followed by 25-HC treatment for 5 h. HMGCR protein was reduced by 80% in cells expressing the WT form of human UBIAD1 after receiving 25-HC treatment. However, 25-HC-induced HMGCR degradation was only reduced by 10% to 50% in cells expressing any of the 21 UBIAD1 mutations ( Fig 3B-3F, top panel), indicating that HMGCR protein can be stabilized by SCD mutants of UBIAD1. Interestingly, the protein levels of SCD-associated UBIAD1 mutants were all substantially increased without being affected by 25-HC, even though the cells were transfected with the same amount of plasmids (100 ng per dish) ( Fig 3B-3F, second panel). The protein levels of UBIAD1 harboring SCD-associated mutations were 1.9 to 9.5-fold more than WT UBIAD1 (Fig 3B-3F, second panel), while the Insig-1 protein had no changes ( Fig 3B-3F, second panel). These results suggest that the SCD mutations of UBIAD1 render HMGCR proteins more resistance to sterol-induced degradation.

UBIAD1s harboring SCD mutations are sequestered in the ER
We next examined the cellular localization of WT UBIAD1 and UBIAD1 harboring SCD mutations in CHO-K1 cells. Immunofluorescence experiments revealed that the ectopically expressed WT UBIAD1 preferentially co-localized with the Golgi marker GM130 (Fig 4). However, the SCD-associated UBIAD1 mutants had a diffused distribution and co-localized with the ER marker calnexin (Fig 4 and S3 Fig).
We further analyzed the distribution of WT and G186R UBIAD1 under different conditions. stimulated HMGCR to bind more WT Ubiad1, and GGOH reduced the interaction between HMGCR and WT Ubiad1. However, the association of HMGCR and G184R mutated Ubiad1 was stronger than WT Ubiad1, and did not response to 25-HC and GGOH treatments (S5B Fig). Therefore, the SCD-associated mutations may impair ER-to-Golgi transportation of UBIAD1 and enhance UBIAD1-HMGCR interaction to prevent Insig-mediated degradation of HMGCR.

Generation and characterization of Ubiad1 G184R/+ mice
Although many missense mutations of UBIAD1 have been found in SCD patients, the causal relationship between these two remains to be proved. To further investigate the consequence of SCD-associated mutation of UBIAD1 in vivo, we generated Ubiad1 G184R (corresponding to G186R in human) knock-in mice using a knockout-first conditional ready strategy as shown in Fig 5A. The targeted allele was knocked out and conditional ready, and the mice were first crossed with Flp recombinase-expressing strain to generate the floxed allele that expressed WT Ubiad1. The mice were then crossed with EIIA-Cre recombinase transgenic mice to get the whole-body knock-in mice ( Fig 5A). The WT mice had a single band of 400 bp, while the heterozygous knock-in mice (Ubiad1 G184R/+ ) exhibited bands at both 400 bp and 500 bp ( Fig 5B). Further sequencing of these two bands confirmed that the mutated band indeed carried the GGA-to-AGA mutation ( Fig 5C).
No homozygous knock-in (Ubiad1 G184R/G184R ) mice were generated when the heterozygotes were intercrossed (Fig 5D). These results are not surprising as Ubiad1 knockout mice defective in vitamin K 2 synthesis are embryonic lethal as well [12]. The WT and heterozygous knock-in mice (Ubiad1 G184R/+ ) were born at an expected Mendelian ratio (34% vs 66%) ( Fig 5D). Ubi-ad1 G184R/+ knock-in mice appeared indistinguishable from WT littermates, and both had similar body weights even at the average age of 105-week-old ( Fig 5E). Given that systemic hypercholesterolemia has been reported in some but not all SCD patients [1], we next sought to evaluate cholesterol levels of WT and Ubiad1 G184R/+ mice. The serum levels of total cholesterol (TC), high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) cholesterol in Ubiad1 G184R/+ knock-in mice were similar to those of WT mice (Fig 5F, 5G and 5H). The serum triglyceride (TG) level of Ubiad1 G184R/+ mice was slightly, but not significantly, decreased relative to WT mice ( Fig 5I). Collectively, these results suggest that the G184R mutation of Ubiad1 does not affect systemic cholesterol and triglyceride levels.

Accumulation of free cholesterol in the cornea of aged Ubiad1 G184R/+ mice
Next, we examined mouse eyes using stereomicroscope. Prominent signs of corneal opacifications were found in 64% (21/33) of both male (11/17) and female (10/16) Ubiad1 G184R/+ mice at 102-to-108 weeks of age (about 2 years, equivalent to the human age of 70) (Fig 7A, bottom  panel). There was no difference between male and female mice. These corneal opacifications were washed with PBS, and depleted of sterols in medium containing 5% LPDS, 1 μM lovastatin and 50 μM mevalonate. Following depletion for 16 hr, cells were treated with or without 1 μg/ml 25-HC and 10 mM mevalonate for 6 hr. Cells were harvested for immunoblotting with indicated antibodies. Actin was a loading control. The relative protein levels of HMGCR and UBIAD1 were quantified with Image-Pro Plus 6 software, then were normalized to the Actin loading control. The normalized intensities of HMGCR without 25-HC and mevalonate treatment were defined as 1. For UBIAD1 proteins, the normalized intensities of WT UBIAD1 bands were defined as 1, and the average intensity of two UBIAD1 bands (with or without 25-HC) are shown. The experiments are repeated three times and representative data are shown. https://doi.org/10.1371/journal.pgen.1008289.g003 were haze-like and quite similar to human SCD (Fig 7A, bottom panel). As controls, none of the 17 aged WT littermates showed corneal opacification (Fig 7A, top panel). To further characterize this corneal opacification, the corneal section from these aged mice were stained with Filipin, a specific antibiotic binding to free cholesterol [32]. Free cholesterol in the WT cornea mainly localized to the epithelial cells and sporadically to the stromal cells as well (Fig 7B,  top panel). However, in Ubiad1 G184R/+ cornea the Filipin signals infiltrated throughout the anterior stroma underneath the epithelium in puncta or patches (Fig 7B, bottom panel), which correspond to the cholesterol crystals observed in the corneas of human SCD patients. Biochemical analysis showed that Ubiad1 G184R/+ mice had higher levels of total and free cholesterol in the cornea than WT littermates, although both showed similar corneal TG levels ( Fig  7C-7E). In contrast, the levels of total cholesterol, free cholesterol and TG levels in the liver, pancreas, lung and spleen were similar between WT and Ubiad1 G184R/+ mice (S7A- S7L Fig).
Collectively, these results demonstrate that the G184R mutation of Ubiad1 specifically cause free cholesterol accumulation in the anterior stroma of cornea, phenocopying human SCD.

Discussion
Based on the above findings, we propose a working model depicting how UBIAD1 mutations cause cholesterol accumulation in the cornea (Fig 8). Under normal conditions in which the WT form of UBIAD1 mainly localizes in the Golgi, an elevation in sterols triggers HMGCR binding to Insig-1 or Insig-2, which recruits E3 ubiquitin ligases including gp78, TRC8 and RNF145 for ubiquitination and proteasomal degradation of HMGCR [18][19][20][21][22]. SCD-associated mutations of UBIAD1 impair its transportation from the ER to Golgi, resulting in a more stable protein in the ER that competes with Insig-1 for binding to HMGCR. As a consequence, sterol-induced ubiquitination and degradation of HMGCR is blocked. The increased cholesterol biosynthesis eventually causes cholesterol accumulation in the cornea in aged mice and humans.
The association of UBIAD1 with HMGCR was recently reported by Debose-Boyd and coworkers using HMGCR as a bait [26]. They showed that the SCD-associated mutants of UBIAD1 were sequestered in the ER and protected HMGCR from degradation, leading to the accumulation of HMGCR and cholesterol [26,33]. They also found cholesterol accumulation in the cornea of aged Ubiad1 N100S/N100S mice, which is another SCD model [9]. Our results and theirs are largely consistent and both support a role of HMGCR-UBIAD1 interaction in HMGCR stabilization. However, we employed an unbiased mass spectrometry analysis of immunoprecipitated UBIAD1 and constructed a different knock-in mouse line (Ubiad1 G184R/ + ). Considering that all SCD patients are heterozygotes of UBIAD1 mutation, our Ubiad1 G184R/ + mouse model may mimic human situation more closely. Indeed, Ubiad1 G184R/+ mice displayed HMGCR accumulation in multiple tissues and elderly ones had free cholesterol deposition in the cornea.
Notably, corneal opacification and free cholesterol accumulation, albeit prominent in twoyear-old Ubiad1 G184R/+ heterozygote mice, were barely detected in the 3-month-old mice (data not shown). Consistently, SCD patients carrying heterozygous UBIAD1 mutations display slow progression of corneal opacification with aging. In addition, the homozygous Ubi-ad1 G184R/G184R knock-in mice are embryonic lethal, while Ubiad1 N100S/N100S mice are grossly  normal. Because Ubiad1 knockout mice fail to survive through development owing to the defects in vitamin K 2 synthesis [12], it is reasonable to speculate that the Ubiad1 G184R mutation may affect vitamin K 2 synthesis more profoundly than Ubiad1 N100S.
Another intriguing phenomenon is that no obvious differences were detected in the serum and tissue levels of cholesterol and triglyceride between the WT and Ubiad1 G184R/+ mice ( Fig  5, Fig 7, S7 Fig), though HMGCR protein levels in all examined tissues were increased (Fig 6). Cholesterol accumulation was only detected in the cornea (Fig 7). Such phenomena are likely attributed to the unique anatomic structure of cornea, which lacks blood-vascular system and is separated from the systemic circulation [34]. Other tissues such as the liver, pancreas, lung and spleen, can intensively exchange lipids with the systemic circulation. In addition, compensatory mechanisms, such as the cholesterol esterification and efflux, may balance the cholesterol level in these tissues. According to the BioGPS expression database, the expression of acyl-CoA: cholesterol acyltransferases (ACAT)-1 and ACAT-2, which convert free cholesterol to cholesteryl ester for storage or secretion as lipoproteins [35], and ABCA1, which is an essential transporter for cholesterol efflux to HDL [36], is very low in the cornea [37]. Thus, the cornea cannot efficiently remove excess cholesterol by converting to cholesteryl ester or pumping out of the cell, leading to the accumulation of free cholesterol once HMGCR is stabilized by UBIAD1 mutations. Interestingly, familial lecithin-cholesterol acyltransferase (LCAT) deficiency and fish eye disease caused by partial loss-of-function of LCAT also exhibit evident corneal opacification due to free cholesterol accumulation [38]. These diseases highlight the importance of cholesterol homeostasis in maintaining corneal function.
Currently, corneal transplant surgery is the only way to restore vision in SCD patients [1]. It is urgent to develop other treatments in the future. As corneal overproduction of cholesterol is caused by HMGCR accumulation, it would be interesting to test whether local application of statins, the well-known inhibitors of HMGCR and are widely used for lowering cholesterol [39], in the form of eyedrops is effective for reducing corneal opacification. In addition, 2-hydroxypropylβ-cyclodextrin (HPβCD) has an excellent ability to solubilize cholesterol and has been used to treat Niemann-Pick type C (NPC) disease caused by lysosomal cholesterol accumulation [40]. The local application of HPβCD would be another promising strategy to reduce corneal cholesterol. Our recently identified HMG499 (also named Cmpd 81) might also be used to treat SCD since HMG499 is a potent HMGCR degrader [25] and SCD is caused by HMGCR stabilization.
Collectively, we have found that the SCD-associated mutants of UBIAD1 bind and stabilize HMGCR, thereby increasing cellular cholesterol level. We have also generated and characterized a mouse model (Ubiad1 G184R/+ ) for SCD disease that will be valuable for studying the underlying mechanism of SCD and developing therapeutic strategies as well.

Ethics statement
All procedures and care of animals were carried out in accordance with the guidelines and protocols approved by the Institutional Animal Care and Use Committee at the Wuhan University under protocol number WDSKY0201408. from the gel, and extracted with DNA gel extraction kit, then sequenced with P1 primer. Sequencing peak maps were visualized by Chromas software. Arrow and box indicated the G184R mutated site. (D) Breeding data of Ubiad1 G184R/+ heterozygote intercrosses. (E-I) Male aged (102-week to 108-week old) WT (6-8 mice/group) and Ubiad1 G184R/+ littermates (13-17 mice/group) were fed ad libitum with chow diet before sacrifice. Blood were harvested and subjected to the following analyses. Values are means ± SD; p value was calculated with Student's t test; ns, no significance.  and delipidated-fetal calf serum (FCS) [42] was prepared from FCS (S1580, Biowest) by ultracentrifugation in our laboratory [25].

Plasmids
The following plasmids pCMV-Insig-1-Myc, pCMV-HMGCR-T7, pCMV-HMGCR-T7-K89R/K248R, pEF1a-HA-Ubiquitin were constructed in our laboratory [25]. The coding regions of UBIAD1 was amplified from human cell cDNA or mouse liver cDNA using standard PCR and cloned into cloned into pcDNA3-C-5xMyc vector. The plasmids encoding the variants of UBIAD1 were generated by site-directed mutagenesis based on full-length human or mouse UBIAD1. All the constructs were verified by DNA sequencing. The primer sequences are listed in S2 Table. Cell culture HEK-293 cells were grown in monolayer at 37˚C in 5% CO 2 in medium containing 10% FCS, Dulbecco's modified Eagle's medium (DMEM), 100 units/ml penicillin and 100 μg/ml streptomycin sulfate. CHO-K1 cells were grown in monolayer at 37˚C in 5% CO 2 in medium containing 5% FCS, Ham's F-12 and DMEM (1:1), 100 units/ml penicillin and 100 μg/ml streptomycin sulfate.

Transient transfection of CHO-K1 Cells
On day 0, CHO-K1 cells were set up for experiments at 4×10 5 cells per 60-mm dish. On day 2, the cells were transfected with the indicated plasmids by using FuGENE HD reagent. The total amount of DNA in each transfection was adjusted to 2 μg per dish by addition of pcDNA3 mock vector.

Membrane fraction extraction
About 50 mg tissues excised from mouse were suspended in 500 μl of buffer A (10 mM HEPES/ KOH, pH 7.6, 1.5 mM MgCl 2 , 10 mM KCl, 5 mM EDTA,5 mM EGTA, 250 mM sucrose) containing protein protease inhibitors, and homogenized by Precellys 24 (Bertin). The homogenized suspensions were then passed through a #7 needle for 30 times and centrifuged at 1000 g at 4˚C for 7 min. The supernatant from the 1,000 g spin was centrifuged at 1 x 10 5 g for 30 min at 4˚C. The pellets were the membrane fractions and were dissolved in 0.1 ml of SDS lysis buffer (10 mM Tris-HCl, pH6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, 1 mM EGTA).

Tandem affinity purification coupled mass spectrometry
HEK-293/Hs-UBIAD1-WT-TAP and HEK-293/Hs-UBIAD1-G186R-TAP stable cells were set up in 100-mm dish. Cells were harvested and lysed in immunoprecipitation (IP) buffer (1% digitonin, PBS, 5 mM EDTA, 5 mM EGTA) with protein protease inhibitors. Then cells were needled 15 times and centrifuged at 13,200 rpm for 20 min. The supernatant was pre-cleared with protein A/G agarose (sc-2003, Santa Cruz Biotechnology), then immunoprecipitated with rabbit IgG coupled agarose (A2909, Sigma) at 4˚C for 2 hr. Agarose-captured proteins were released with TEV Protease (P8112S, New England Biolabs) in 500 μl IP buffer at RT for 1 hr. The released proteins were immunoprecipitated with anti-Flag M2 agarose beads (A2220, Sigma) at 4˚C for 2 hr, and eluted with 3x Flag peptide at 4˚C for 30 min. The eluted fractions were resolved with SDS-PAGE and stained with Coomassie blue. The identifies of proteins from eluted fractions were determined by tandem mass spectrometry.

Co-immunoprecipitation
The cells were harvested and suspended in 600 μl of IP buffer (1% digitonin, PBS, 5 mM EDTA, 5 mM EGTA, and 10 mM N-Ethylmaleimide) containing protein protease inhibitors, and passed through #7 needle 15 times. The cell lysates were centrifuged at 13,200 rpm at 4˚C for 10 min, immunoprecipitated with anti-T7 antibody coupled agaroses (69026, Merck), and eluted with 2x SDS loading buffer. Whole cell lysates and pellets were subjected to SDS-PAGE and immunoblotted with indicated antibodies.

Immunofluorescence staining
Cells were fixed with 4% PFA in PBS, permeabilized with 0.2% Triton X-100 (T8787, Sigma) for 8 minutes, blocked with 2% BSA for 1 hr and incubated with indicated primary antibodies for 1 hr at room temperature. Fluorescence-labeled secondary antibodies were used at concentration of 4 μg/ml in PBS containing 0.2% BSA for 45 minutes. Nuclei was stained with Hoechst 33342. Immunofluorescence images were captured by Leica TCS SP2 confocal microscope. Images were acquired at identical laser output, gain, and offset [44].

Filipin staining
Corneas were fixed by 4% PFA and sectioned with frozen cryostat (Leica CM3050 S) at 7 μm. Frozen sectioned slides were washed twice with PBS, stained with 50 μg/ml filipin in 10% FBS/ PBS for 1 hr at room temperature, washed three times with PBS and mounted. Images were captured by fluorescence microscopy (Zeiss Axio Imager Z2) using a UV filter set, and the intensity of mercury lamp was turned to 10% of the maximal strength. Images were acquired at identical output, gain, and offset [46].

Analysis of de novo synthesis of lipid
Cells were incubated in medium containing 10% delipidated-FCS, 1 μM lovastatin and 50 μM mevalonate for 16 hr. After depletion, cells were washed to remove lovastatin, and change to medium with 10% delipidated -FCS for 3 hr. Then [ 14 C]-acetate (36 μCi/100-mm dish) were added and cells were treated for additional 2 hr. Cells were washed twice with PBS, dissolved by 0.5 ml 0.1 N NaOH, and saponified with ethanol and 75% potassium hydroxide for 2 hr. Then the nonpolar lipids (cholesterol) were extracted in petroleum ether and evaporated to dryness with N 2 . Following addition of concentrated HCl, polar lipids (fatty acids) were extracted in petroleum ether and evaporated to dryness with N 2 . The lipids were resolved by thin-layer chromatography (1.05582.0001, Merck). Radioactive signals were visualized with phosphoimager, and images were captured with Typhoon FLA 9000 (GE Healthcare).

Generation of Ubiad1 G184R/+ mice
The knockout first conditional ready targeting vector, containing a gene-trap cassette, was electroporated into ES cell line from C57BL/6J mice. Positive ES clones were verified by PCR and Southern blot, and three positive ES clones were injected to BALB/c blastocysts to generate chimeric mice. Male mice with high percentage chimeras were bred with female C57BL/6J mice (Shanghai Laboratory Animal Company, China) to get the heterozygous Ubiad1 knockout mice. To get a conditional knockin allele, these heterozygous knockout mice were bred with Flp recombinase deleter transgene (Jackson Laboratory, USA) to remove the Frt-flanked cassette. Subsequently, these mice were crossed with EIIA-Cre recombinase transgenic mice (Jackson Laboratory, USA) to remove Loxp-flanked cassette, thus getting the whole tissue Ubiad1 G184R knockin mice. The genotypes were identified by PCR using primers P1 (GCAAGCTGTATTTTGCCTTG), and P2 (CGAAAGTGATGAGGATGACGAGGT). The male and female Ubiad1 G184R/+ were intercrossed to get Ubiad1 +/+ and Ubiad1 G184R/+ mice littermates for experiments. All mice were maintained on a 12-h light/dark schedule, and fed ad libitum access to water and standard chow diet (Shanghai Laboratory Animal Company, China).

Biochemistry analyses of lipids in serum and tissue
Blood were collected from anesthetized mice, and serum was prepared from blood by centrifuging at 1500 × g for 10 min. Tissues were excised and weighted, then homogenized in chloroform/methanol (2:1) with Precellys 24 (Bertin). The lipids were extracted and dried under N 2 , and dissolved in ethanol. Total cholesterol and free cholesterol levels in serum and tissues were determined with Amplex Red Cholesterol Assay Kit (A12216, Invitrogen). HDL cholesterol, LDL cholesterol and triglyceride levels in the serum and liver were measured with HDL cholesterol, LDL cholesterol and triglyceride Assay Kit (Shanghai Kehua Bio-engineering, China), respectively.

Generation of mouse embryonic fibroblasts (MEFs)
Female Ubiad1 G184R/+ mice were mated with male Ubiad1 G184R/+ mice. Pregnant mice on the 13-day of conception were sacrificed by brief exposure to CO 2 . The uterine horns were excised into Petri dish and all embryos were carefully dissected. Each embryo was transferred into a new dish; the heads were used for genotyping. The rest of embryo was minced thoroughly using sharp Iris scissors, and digested with trypsin/EDTA for 20 minutes. Trypsin was neutralized, and the MEFs were collected by centrifuging. MEFs were cultured in medium with 10% FCS/DMEM, 100 units/ml penicillin and 100 μg/ml streptomycin sulfate.

Corneal examinations
Mice were first anaesthetized with 1% pentobarbital sodium in PBS (10 μl/g), then corneal opacifications were observed and images were captured with stereoscopic microscope (Olympus SZX16). The corneas were excised from eyes and carefully removed other tissues under stereomicroscope, then used for protein and lipid analyses.

Statistical analysis
The statistical analyses were carried out using GraphPad Prism 6 software. Data were presented as means ± SD and analyzed by unpaired two-tailed Student's t-test. Statistical significance was set at p < 0.05.  Table. (TIF) S2 Fig. Amino acids of SCD-associated mutations in UBIAD1 are evolutionarily conserved ranging from fly to human. The protein sequences of UBIAD1 from 16 species were aligned using ClustalW algorithm in MEGA X software. These species include human, chimpanzee, rhesus, tree shrew, sheep, horse, elephant, dog, rabbit, mouse, chicken, snake, frog, zebrafish, sea urchin and fly. Evolutionary conserved residues are marked with asterisk, and SCD-associated mutations are marked with red. Values are means ± SD; p value was calculated with Student's t test; ns, no significance. The levels of total cholesterol (A), free cholesterol (B), and triglyceride (C) in liver from WT and Ubiad1 G184R/+ mice. The levels of total cholesterol (D), free cholesterol (E), and triglyceride (F) in pancreas from WT and Ubiad1 G184R/+ mice. The levels of total cholesterol (G), free cholesterol (H), and triglyceride (I) in lung from WT and Ubi-ad1 G184R/+ mice. The levels of total cholesterol (J), free cholesterol (K), and triglyceride (L) in spleen from WT and Ubiad1 G184R/+ mice. The experiments are repeated three times and representative data are shown. (TIF) S1