Regulated degradation of HMG CoA reductase requires conformational changes in sterol-sensing domain.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2-4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2-6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations-one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.

Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol.

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

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

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.

Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

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

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.

Phosphatidylserine transport to the mitochondria is regulated by ubiquitination.

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.