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• W2343171047 abstract "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. 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. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) belongs to the UbiA superfamily of integral membrane prenyltransferases (1Heide L. Prenyl transfer to aromatic substrates: genetics and enzymology.Curr. Opin. Chem. Biol. 2009; 13: 171-179Crossref PubMed Scopus (146) Google Scholar). These enzymes contain 8–10 transmembrane helices and catalyze transfer of isoprenyl groups to aromatic acceptors, producing a wide range of molecules such as ubiquinones, hemes, chlorophylls, vitamin E, and vitamin K. In animals, UBIAD1 catalyzes transfer of the 20-carbon geranylgeranyl moiety from geranylgeranyl pyrophosphate (GGpp) to menadione released from plant-derived phylloquinone, thereby generating the vitamin K2 subtype, menaquinone-4 (MK-4) (2Nakagawa K. Hirota Y. Sawada N. Yuge N. Watanabe M. Uchino Y. Okuda N. Shimomura Y. Suhara Y. Okano T. Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme.Nature. 2010; 468: 117-121Crossref PubMed Scopus (230) Google Scholar, 3Hirota Y. Tsugawa N. Nakagawa K. Suhara Y. Tanaka K. Uchino Y. Takeuchi A. Sawada N. Kamao M. Wada A. et al.Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.J. Biol. Chem. 2013; 288: 33071-33080Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Mutations in the UBIAD1 gene are associated with Schnyder corneal dystrophy (SCD), an autosomal dominant human eye disease characterized by progressive opacification of the cornea, owing to abnormal accumulation of cholesterol and other lipids (4Orr A. Dube M.P. Marcadier J. Jiang H. Federico A. George S. Seamone C. Andrews D. Dubord P. Holland S. et al.Mutations in the UBIAD1 gene, encoding a potential prenyltransferase, are causal for Schnyder crystalline corneal dystrophy.PLoS One. 2007; 2: e685Crossref PubMed Scopus (106) Google Scholar, 5Weiss J.S. Kruth H.S. Kuivaniemi H. Tromp G. White P.S. Winters R.S. Lisch W. Henn W. Denninger E. Krause M. et al.Mutations in the UBIAD1 gene on chromosome short arm 1, region 36, cause Schnyder crystalline corneal dystrophy.Invest. Ophthalmol. Vis. Sci. 2007; 48: 5007-5012Crossref PubMed Scopus (92) Google Scholar). Systemic dyslipidemia appears to be associated with some, but not all, SCD cases (6Brownstein S. Jackson W.B. Onerheim R.M. Schnyder's crystalline corneal dystrophy in association with hyperlipoproteinemia: histopathological and ultrastructural findings.Can. J. Ophthalmol. 1991; 26: 273-279PubMed Google Scholar, 7Crispin S. Ocular lipid deposition and hyperlipoproteinaemia.Prog. Retin. Eye Res. 2002; 21: 169-224Crossref PubMed Scopus (69) Google Scholar). Missense mutations that alter 20 amino acid residues in UBIAD1 have been identified in ∼50 SCD families (8Nickerson M.L. Bosley A.D. Weiss J.S. Kostiha B.N. Hirota Y. Brandt W. Esposito D. Kinoshita S. Wessjohann L. Morham S.G. et al.The UBIAD1 prenyltransferase links menaquinone-4 [corrected] synthesis to cholesterol metabolic enzymes.Hum. Mutat. 2013; 34: 317-329Crossref PubMed Scopus (58) Google Scholar, 9Nowinska A.K. Wylegala E. Teper S. Lyssek-Boron A. Aragona P. Roszkowska A.M. Micali A. Pisani A. Puzzolo D. Phenotype-genotype correlation in patients with Schnyder corneal dystrophy.Cornea. 2014; 33: 497-503Crossref PubMed Scopus (22) Google Scholar). Several of these altered amino acids reside within the active site of UBIAD1 (10Huang H. Levin E.J. Liu S. Bai Y. Lockless S.W. Zhou M. Structure of a membrane-embedded prenyltransferase homologous to UBIAD1.PLoS Biol. 2014; 12: e1001911Crossref PubMed Scopus (70) Google Scholar, 11Cheng W. Li W. Structural insights into ubiquinone biosynthesis in membranes.Science. 2014; 343: 878-881Crossref PubMed Scopus (93) Google Scholar). A link between UBIAD1 and cholesterol metabolism was first provided by coimmunoprecipitation studies that showed an association of UBIAD1 with the cholesterol biosynthetic enzyme, HMG-CoA reductase (8Nickerson M.L. Bosley A.D. Weiss J.S. Kostiha B.N. Hirota Y. Brandt W. Esposito D. Kinoshita S. Wessjohann L. Morham S.G. et al.The UBIAD1 prenyltransferase links menaquinone-4 [corrected] synthesis to cholesterol metabolic enzymes.Hum. Mutat. 2013; 34: 317-329Crossref PubMed Scopus (58) Google Scholar). More recently, we showed that UBIAD1 inhibits sterol-accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of reductase (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar), one of several feedback mechanisms that converge on the enzyme to maintain cholesterol homeostasis (13Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Scopus (4537) Google Scholar). The polytopic, ER-localized HMG-CoA reductase catalyzes reduction of HMG-CoA to mevalonate, a rate-limiting step in synthesis of cholesterol as well as nonsterol isoprenoids including farnesyl pyrophosphate (Fpp) and GGpp that are transferred to many cellular proteins and utilized in synthesis of ubiquinone, MK-4, heme, and dolichol (2Nakagawa K. Hirota Y. Sawada N. Yuge N. Watanabe M. Uchino Y. Okuda N. Shimomura Y. Suhara Y. Okano T. Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme.Nature. 2010; 468: 117-121Crossref PubMed Scopus (230) Google Scholar, 13Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Scopus (4537) Google Scholar, 14Zhang F.L. Casey P.J. Protein prenylation: molecular mechanisms and functional consequences 3.Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1733) Google Scholar). Intracellular accumulation of sterols causes reductase to bind ER membrane proteins called Insigs (15Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 16Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), which leads to ubiquitination of the enzyme by Insig-associated ubiquitin ligases (16Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 17Song B.L. Sever N. DeBose-Boyd R.A. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase.Mol. Cell. 2005; 19: 829-840Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 18Jo Y. Lee P.C. Sguigna P.V. DeBose-Boyd R.A. Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.Proc. Natl. Acad. Sci. USA. 2011; 108: 20503-20508Crossref PubMed Scopus (110) Google Scholar, 19Liu T.F. Tang J.J. Li P.S. Shen Y. Li J.G. Miao H.H. Li B.L. Song B.L. Ablation of gp78 in liver improves hyperlipidemia and insulin resistance by inhibiting SREBP to decrease lipid biosynthesis.Cell Metab. 2012; 16: 213-225Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Ubiquitinated reductase is then extracted across ER membranes and released into the cytosol for degradation by 26S proteasomes (20Elsabrouty R. Jo Y. Dinh T.T. DeBose-Boyd R.A. Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.Mol. Biol. Cell. 2013; 24: 3300-3308Crossref PubMed Google Scholar, 21Morris L.L. Hartman I.Z. Jun D.J. Seemann J. DeBose-Boyd R.A. Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.J. Biol. Chem. 2014; 289: 19053-19066Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Maximal degradation requires the addition to cells of geranylgeraniol (GGOH), the alcohol derivative of GGpp (16Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). We postulate that GGOH becomes converted to GGpp, which augments reductase ERAD by enhancing its membrane extraction (21Morris L.L. Hartman I.Z. Jun D.J. Seemann J. DeBose-Boyd R.A. Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.J. Biol. Chem. 2014; 289: 19053-19066Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Recently, we found that sterols also cause reductase to bind to UBIAD1 (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar). GGpp triggers release of UBIAD1 from reductase, allowing for its maximal ERAD. Importantly, the addition to cells of farnesol, the alcohol derivative of Fpp, neither inhibits the binding of UBIAD1 to reductase nor augments ERAD of reductase (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar, 16Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Two of the 20 SCD-associated mutants of UBIAD1 (N102S and G177R) resisted GGpp-induced displacement from reductase and thereby blocked its degradation (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar). In the course of our studies, we discovered that GGpp also stimulates translocation of UBIAD1 from the ER to the Golgi (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar). SCD-associated UBIAD1 (N102S) and (G177R) are refractory to this GGpp-induced Golgi transport and localize to the ER. Building on these observations, we show in the current study that UBIAD1 localizes to the medial-trans cisternae of the Golgi in isoprenoid-replete cells. All 20 of the SCD-associated mutants of UBIAD1 are defective in Golgi transport and remain sequestered in the ER where they inhibit reductase ERAD in a seemingly dominant-negative fashion. Intriguingly, acute depletion of isoprenoids triggers rapid retrograde transport of UBIAD1 from the Golgi to the ER. Although UBIAD1 localizes to the Golgi of isoprenoid-replete cells in the steady state, the protein accumulates in the ER when transport from the organelle is blocked. These findings suggest a model in which UBIAD1 constitutively cycles between the Golgi and ER. Upon sensing GGpp depletion in membranes of the ER, UBIAD1 becomes trapped in the organelle and inhibits reductase ERAD so as to stimulate mevalonate synthesis for replenishment of GGpp. This novel sensing mechanism directly controls ERAD of reductase and becomes disrupted in SCD, which likely contributes to the accumulation of cholesterol that characterizes the eye disease. We obtained GGOH, GGpp, Fpp, nocodazole, and brefeldin A (BFA) from Sigma-Aldrich (St. Louis, MO) and Santa Cruz Biotechnology (Dallas, TX); cycloheximide was obtained from Cell Signaling Technology (Danvers, MA); 25-hydroxycholesterol and cholesterol was obtained from Steraloids (Newport, RI); hydroxypropyl β-cyclodextrin was obtained from (Cyclodextrin Technologies Development, Alachua, FL). Recombinant His-tagged Sar1DN was expressed in Escherichia coli and isolated on Ni-NTA agarose (Qiagen, Valencia, CA) as previously described (22Rowe T. Balch W.E. Expression and purification of mammalian Sarl.Methods Enzymol. 1995; 257: 49-53Crossref PubMed Scopus (33) Google Scholar). The buffer was exchanged by dialysis against 25 mM HEPES-KOH (pH 7.2), 125 mM potassium acetate, 1 mM MgCl2, 1 mM glutathione, 10 μM guanosine diphosphate, and 50 μM EGTA. SR-12813 was synthesized by the Core Medicinal Chemistry laboratory at the University of Texas Southwestern Medical Center or obtained from Sigma-Aldrich. Other reagents, including newborn calf lipoprotein-deficient serum (LPDS, d > 1.215 g/ml), sodium compactin, and sodium mevalonate, were prepared or obtained as previously described (20Elsabrouty R. Jo Y. Dinh T.T. DeBose-Boyd R.A. Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells.Mol. Biol. Cell. 2013; 24: 3300-3308Crossref PubMed Google Scholar, 23Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1282) Google Scholar). The expression plasmids, pCMV-Myc-UBIAD1, which encodes human UBIAD1 containing a single copy of a Myc epitope at the N-terminus under transcriptional control of the cytomegalovirus (CMV) promoter, pCMV-Myc-UBIAD1 (N102S) encoding Myc-tagged human UBIAD1 harboring the SCD-associated asparagine-102 to serine (N102S) mutation, and pCMV-Myc-UBIAD1 (G177R) encoding Myc-tagged human UIBAD1 harboring the SCD-associated glycine-177 to arginine mutation were previously described (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar). The remaining SCD-associated mutants of UBIAD1 were generated using the QuikChange® site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and pCMV-Myc-UBIAD1 as a template. The expression plasmid, pDsRed-Golgi, encoding a fusion protein consisting of DsRed-Monomer and the N-terminal 81 amino acids of human β1,4-galactosyltransferase was obtained from Clontech. SV-589 cells are a line of immortalized human fibroblasts expressing the SV40 large T-antigen (24Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (979) Google Scholar). Monolayers of SV-589 cells were maintained in medium A (DMEM containing 1,000 mg/l glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate) supplemented with 10% (v/v) FCS at 37°C, 5% CO2. SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated by transfection of SV-589 cells with 3 μg pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent (Promega, Madison, WI) as described below, followed by 2 weeks of selection in medium A supplemented with 10% FCS and 700 μg/ml G418. Individual colonies were isolated using cloning cylinders. Clonal isolates from expanded colonies were obtained using serial dilution in 96-well plates. Clones were evaluated by immunofluorescence microscopy using IgG-9E10 against the Myc epitope (described below). CHO-K1/pMyc-UBIAD1 and UT-2/pMyc-UBIAD1, lines of CHO-K1 and reductase-deficient UT-2 cells (25Mosley S.T. Brown M.S. Anderson R.G. Goldstein J.L. Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3 -methylglutaryl coenzyme A reductase.J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar) that stably express Myc-UBIAD1, were generated by transfection of cells with 3 μg pCMV-Myc-UBIAD1 as described below, followed by 2 weeks of selection in medium B (1:1 mixture of Ham's F-12 medium and DMEM containing 100 U/ml penicillin and 100 μg/ml streptomycin sulfate) containing 5% FCS and 700 μg/ml G418. The medium for UT-2 cells was further supplemented with 200 μM mevalonate. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescence using IgG-9E10 as described below. CHO-K1/pMyc-UBIAD1 cells were maintained in monolayer in medium B containing 5% FCS and 700 μg/ml G418 at 37°C, 8% CO2. Monolayers of UT-2/pMyc-UBIAD1 cells were grown at 37°C, 8% CO2 in identical medium supplemented with 200 μM mevalonate. CHO-7 cells, a subline of CHO-K1 cells selected for growth in LPDS (26Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells.J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar), were grown in monolayer at 37°C, 8% CO2. The cells were maintained in medium B supplemented with 5% LPDS. Monolayers of SRD-13A/pGFP-Scap, a line of Scap-deficient CHO-7 cells that stably express GFP-Scap (27Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), were maintained at 37°C, 8% CO2 in medium B supplemented with 5% LPDS. Transient transfection of cells with FuGENE6 transfection reagent was carried out as previously described (12Schumacher M.M. Elsabrouty R. Seemann J. Jo Y. DeBose-Boyd R.A. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.eLife. 2015; 4: 05560Crossref Scopus (61) Google Scholar, 15Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain.Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Conditions of subsequent incubations are described in the figure legends. Following incubations, cells were washed with PBS and subsequently fixed and permeabilized for 15 min in methanol at −20°C. Upon blocking with 1 mg/ml BSA in PBS, coverslips were incubated for 1 h at 37°C with primary antibodies [IgG-H8, a mouse monoclonal antibody against human UBIAD1 (Santa Cruz Biotechnology), rabbit polyclonal anti-GM130 IgG (28Diao A. Rahman D. Pappin D.J. Lucocq J. Lowe M. The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.J. Cell Biol. 2003; 160: 201-212Crossref PubMed Scopus (199) Google Scholar), IgG-9E10, a mouse monoclonal antibody against c-Myc purified from the culture medium of hybridoma clone 9E10 (American Type Culture Collection, Manassas, VA), and rabbit polyclonal anti-TGN46 (Abcam, Cambridge, MA)] diluted in PBS containing 1 mg/ml BSA. Bound antibodies were visualized with goat anti-mouse IgG conjugated to Alexa Fluor 488 or Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 594 (Life Technologies, Grand Island, NY) as described in the figure legends. Coverslips were also stained for 5 min with 300 nM 4′,6-diamidino-2-phenylindole (Life Technologies) to visualize nuclei. The coverslips were then mounted in Mowiol 4-88 solution (Calbiochem/EMD Millipore, Billerica, MA) or Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Fluorescence imaging was performed using a DeltaVision microscopy imaging system (GE Healthcare Life Sciences, Pittsburgh, PA) equipped with a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ) and objective oil lenses 60×/1.42 and 100×/1.40 (Olympus, Waltham, MA) as indicated in figure legends. The z stacks were deconvolved and Pearson correlation coefficients for each z stack were generated using DeltaVision SoftWoRx software (GE Healthcare Life Sciences, Pittsburgh, PA). Additional fluorescence imaging was performed using a Zeiss Axio Observer Epifluorescence microscope using a 63×/1.4 oil plan-apochromat objective and Zeiss Axiocam color digital camera (Zeiss, Peabody, MA) in black and white mode as indicated in the figure legends. Brightness levels were adjusted across the entire images using ImageJ software (National Institutes of Health, Bethesda, MD). Rat liver cytosol was prepared from adult male Sprague-Dawley rats as previously described (29Song B.L. DeBose-Boyd R.A. Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase.J. Biol. Chem. 2004; 279: 28798-28806Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Cytosol from isoprenoid-depleted UT-2 cells was prepared as follows. UT-2 cells were set up on day 0 at 3 × 105 cells per 100 mm dish in medium B containing 5% FCS and 200 μM mevalonate. On day 3, cells were depleted of isoprenoids though incubation in medium B supplemented with 5% FCS in the absence of mevalonate. After 16 h at 37°C, cells were harvested into the medium by scraping and collected by centrifugation, after which cell pellets were washed with PBS supplemented with a protease inhibitor cocktail consisting of 20 μM leupeptin, 5 μg/ml pepstatin A, 2 μg/ml aprotinin, 25 μg/ml N-acetyl-leucinal-leucinal-norleucinal, and 1 mM dithiotheitol. The washed cells from 250 dishes were resuspended in buffer containing 50 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 70 mM potassium acetate, 5 mM potassium EGTA, and 2.5 mM magnesium acetate supplemented with protease inhibitors. The resuspended cells were then lysed by passage through an 18 gauge needle 30 times, followed by 25 strokes in a 50 ml Dounce homogenizer fitted with a Teflon pestle. The resulting homogenates were subjected to centrifugation at 1,000 g for 10 min at 4°C. The supernatant of this spin was then subjected to centrifugation at 100,000 g for 1 h at 4°C. The final supernatant, designated as cytosol (∼2 mg/ml), was divided into multiple aliquots, snap-frozen in liquid nitrogen, and stored at −80°C. For experiments, tubes were thawed in a 37°C water bath and placed on ice until use. SV-589 cells were set up for experiments as described in the figure legends. Following incubations described in figure legends, cells were harvested into medium by scraping and pooled suspensions from triplicate dishes were subjected to centrifugation at 1,000 g for 5 min at 4°C. Cell pellets were resuspended in 0.5 ml buffer containing 10 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 10 mM potassium acetate, 1.5 mM magnesium acetate, and protease inhibitors, passed through a 22 gauge needle 30 times, and centrifuged at 1,000 g for 5 min at 4°C. The resulting supernatants were transferred to siliconized microfuge tubes and centrifuged at 16,000 g for 3 min at 4°C. Each pellet was suspended in 50 μl of buffer containing 50 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 70 mM potassium acetate, 5 mM potassium EGTA, 2.5 mM magnesium acetate, and protease inhibitors to obtain microsomes that were pooled and used in the in vitro vesicle-formation assay described below. The protein concentration of an aliquot (5 μl) of microsomal suspensions was determined using PierceTM Coomassie Plus protein assay reagent (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions using BSA as standard. The protocol used in this study was adapted from previously described procedures (27Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 30Rexach M.F. Schekman R.W. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.J. Cell Biol. 1991; 114: 219-229Crossref PubMed Scopus (229) Google Scholar, 31Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI.J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (150) Google Scholar). In a final volume of 100 μl, each reaction contained 50 mM HEPES-KOH (pH 7.2), 250 mM sorbitol, 70 mM potassium acetate, 5 mM potassium EGTA, 2.5 mM magnesium acetate, 1.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, 4 units/ml of creatine kinase, protease inhibitors, 80–100 μg protein of SV-589 microsomes, and 10–100 μg rat liver or UT-2 cytosol. Incubations were carried out in siliconized 1.5 ml microfuge tubes for 20 min at 37°C (unless otherwise indicated). Reactions were terminated by transfer of tubes to ice, followed by centrifugation at 16,000 g for 3 min at 4°C to obtain a medium-speed pellet (P16) and a medium-speed supernatant (S16). The S16 fraction was then subjected to an additional round of centrifugation at 100,000 g for 30 min at 4°C to obtain a high-speed pellet (P100). The P16 and P100 were resuspended in buffer containing 10 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 1% (w/v) SDS plus protease inhibitors supplemented with 4× SDS-PAGE loading buffer and heated at 95°C for 5 min. Aliquots of the P16 (7.5 μl) and P100 (30 μl) were subjected to 10% SDS-PAGE, transferred to nylon filters, and analyzed by immunoblotting. The P16 and P100 fractions are referred to membranes and vesicles, respectively. Primary antibodies used for immunoblot analysis include: IgG-H8 against human UBIAD1; IgG-4H4, a mouse monoclonal antibody against hamster Scap (32Zhang Y. Motamed M. Seemann J. Brown M.S. Goldstein J.L. Point mutation in luminal loop 7 of Scap protein blocks interaction with loop 1 and abolishes movement to Golgi.J. Biol. Chem. 2013; 288: 14059-14067Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); and rabbit polyclonal anti-ribophorin I IgG (Abcam). Microinjection was performed with a Transjector 5246 and a Micromanipulator 5171 (Eppendorf, Hauppauge, NY). SV-589/pMyc-UBIAD1 cells on glass coverslips were chang" @default.
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• W2343171047 title "Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi" @default.
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