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

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.

Keywords: UbiA prenyltransferase domain-containing protein-1; endoplasmic reticulum; isoprenoid; lipid metabolism; protein trafficking; vitamin K.

Fig. 1.

Kinetics of geranylgeranyl-induced transport of UBIAD1 from the ER to the Golgi. SV-589 (A, B) and SV-589/pMyc-UBIAD1 (C) cells were set up on day 0 at 8 × 10⁴ cells per well of 6-well plates with glass coverslips in medium A containing 10% FCS. On day 1, cells were depleted of isoprenoids though incubation in medium A containing 10% LPDS, 10 μM sodium compactin, and 50 μM sodium mevalonate. After 16 h at 37°C, cells were refed medium A containing 10% LPDS, 10 μM compactin, and 30 μM GGOH. In (B), some of the cells also received 50 μM cycloheximide. Following incubation for the indicated period of time (A, C) or 4 h (B) at 37°C, cells were fixed and analyzed by immunofluorescence microscopy using IgG-H8 (against endogenous UBIAD1) and IgG-9E10 (against transfected Myc-UBIAD1) using a Zeiss Axio Observer Epifluorescence microscope as described in the Materials and Methods. Scale bar is 10 microns. D: CHO-7/pGFP-Scap cells were set up on day 0 at 1 × 10⁵ cells per well of 6-well plates with glass coverslips in medium B supplemented with 5% LPDS. On day 1, cells were transfected with pCMV-Myc-UBIAD1 (1 μg per dish) as described in the Materials and Methods. Four hours after transfection, cells received a direct addition of medium B containing either 5% FCS (panels 1 and 2) or 5% LPDS (panels 3 and 4) (final concentrations). Following incubation for 16 h at 37°C, the dishes received medium B containing either 5% FCS, 1% hydroxypropyl β-cyclodextrin (HPCD), 10 μg/ml cholesterol, and 10 μg/ml 25-hydroxycholesterol (panels 1 and 2) or 5% LPDS, 1% HPCD, and 10 μM compactin (panels 3 and 4). After 6 h at 37°C, cells were fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) using a DeltaVision imaging system as described in Materials and Methods. Scale bar is 5 microns.

Fig. 2.

UBIAD1 localizes to medial-trans region of the Golgi apparatus as determined by deconvolution microscopy. SV-589/pMyc-UBIAD1 cells were set up on day 0 at 1.5 × 10⁵ cells per 60 mm dish with glass coverslips in medium A containing 10% FCS. On day 1, cells were transfected with pDsRed-Golgi (3 μg per dish) as described in the Materials and Methods. Four hours after transfection, cells received a direct addition of medium A containing 10% FCS (final concentration). Following incubation for 16 h at 37°C, cells were treated for 2 h in the absence or presence of 5 μg/ml nocodazole. Cells were then fixed and analyzed by immunofluorescence deconvolution microscopy using IgG-9E10 (against transfected Myc-UBIAD1), anti-GM130, anti-TGN46, and a 100× oil objective as described in the Materials and Methods. Pearson correlation coefficients for the indicated protein and Myc-UBIAD1 are shown in graph below the images.

Fig. 3.

GGpp stimulates incorporation of UBIAD1 into vesicles in vitro. On day 0, SV-589 cells were set up at 2.5 × 10⁵ cells per 100 mm dish in medium A containing 10% FCS. On day 3, cells were switched to medium A containing 10% LPDS, 10 μM compactin, and 50 μM mevalonate. Following incubation for 16 h at 37°C, cells were harvested for preparation of microsomes as described in the Materials and Methods. A–C: Aliquots of pooled microsomes were incubated at the indicated temperature (A) or 37°C (B, C) in the absence or presence of rat liver cytosol (0–25 μl per reaction), ATP, GTP, and an ATP-regenerating system as described in the Materials and Methods. Following incubation for 20 min (A, C) or the indicated period of time (B) at 37°C, reactions were centrifuged to separate vesicles and membranes that were subjected to SDS-PAGE, followed by immunoblot analysis with IgG-H8 (against UBIAD1), IgG-4H4 (against Scap), and anti-ribophorin I. D, E: Aliquots of pooled microsomes were incubated at 37°C with ATP, GTP, and an ATP-regenerating system in the absence or presence of cytosol (0–40 μl per reaction) prepared from reductase-deficient UT-2 cells and the indicated concentration of GGpp or Fpp. After 20 min at 37°C, membranes and vesicles were separated by centrifugation and analyzed by immunoblot with IgG-H8 (against UBIAD1).

Fig. 4.

SCD-associated mutants of UBIAD1 are defective in trafficking from the ER to the Golgi. SV-589 cells were set up on day 0 and transfected on day 1 with 3 μg pCMV-Myc-UBIAD1 (wild-type or indicated mutant) as described in the legend to Fig. 2. Four hours after transfection, cells received a direct addition of medium A containing 10% FCS (final concentration). After 16 h at 37°C, cells were fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) as described in the legend to Fig. 1.

Fig. 5.

SCD-associated mutants of UBIAD1 confer resistance of CHO-7 cells to growth in the presence of SR-12813. CHO-7 cells were set up on day 0 at 4 × 10⁵ cells per 100 mm dish in medium B containing 5% FCS. On day 1, cells were transfected with 3 μg pCMV-Myc-UBIAD1 (wild-type or indicated mutant) as described in the legend to Fig. 2. All of the plasmids contained the G418 resistance gene, neo. On day 2, the cells were switched to medium B containing 5% FCS and 0.7 mg/ml G418 and refed every 2–3 days. On day 14, the G418-resistant cells were trypsinized, washed in medium B containing 5% LPDS, and plated in triplicate at 1 × 10³ cells per well of a 96-well plate containing medium B supplemented with 5% LPDS and the indicated concentration of SR-12813. The cells were refed every 2–3 days. On day 28, the wells were washed with PBS, fixed in 95% ethanol, and stained with crystal violet. The stained plates were scanned on an Epson Perfection V700 photo scanner (Long Beach, CA) in transmitted light mode at a resolution of 300 dots per inch. Images were analyzed using National Institutes of Health Image J software to determine the average pixel value from a circular area corresponding to each well. The resulting values were subtracted from the value obtained from an empty well that received no cells to yield arbitrary units corresponding to degree of growth of each condition tested.

Fig. 6.

Depletion of nonsterol isoprenoids triggers retrograde transport of UBIAD1 from the Golgi to the ER. SV-589 (A, B) and SV-589/pMyc-UBIAD1 cells (C) were set up on day 0 at 8 × 10⁴ cells per well of 6-well plates with glass coverslips in medium A supplemented with 10% FCS. On day 1, cells were refed identical medium; some of the cells in (C) were switched to medium A containing 10% LPDS. After 16 h at 37°C, cells were incubated in the identical medium for the indicated time (A), 6 h (B), or 2 h (C) in the presence of 10 μM compactin; some of the dishes in (B) also received 50 μM cycloheximide. Following treatments, cells were fixed and analyzed by immunofluorescence microscopy using IgG-H8 (against endogenous UBIAD1) and IgG-9E10 (against Myc-UBIAD1) as described in the legend to Fig. 1. Scale bar is 10 microns.

Fig. 7.

HMG-CoA reductase is not required for geranylgeranyl-mediated transport of UBIAD1 between the ER and the Golgi. CHO-K1/pMyc-UBIAD1 and UT-2/pMyc-UBIAD1 cells were set up on day 0 at 8 × 10⁴ cells per well of 6-well plates with glass coverslips in medium B supplemented with 5% FCS; medium for UT-2/pMyc-UBIAD1 cells was further supplemented with 200 μM mevalonate. On day 1, cells were refed medium B containing 5% FCS (no mevalonate). After 16 h at 37°C, cells were treated for 4 h at 37°C with identical medium in the absence or presence of 10 μM compactin and 30 μM GGOH. Following treatments, cells were fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) as described in the legend to Fig. 1. The scale bar is 5 microns.

Fig. 8.

BFA triggers relocalization of UBIAD1 from the Golgi to the ER. SV-589 and SV-589/pMyc-UBIAD1 cells were set up on day 0 at 8 × 10⁴ cells per well of 6-well plates with glass coverslips in medium A containing 10% FCS. On day 1, cells were refed identical medium in the absence or presence of 2 μg/ml BFA. Following incubation at 37°C for the indicated period of time (A) or 0.5 h (B), cells were fixed for immunofluorescence microscopy using IgG-H8 (against endogenous UBIAD1), IgG-9E10 (against Myc-UBIAD1), and anti-TGN46 as described in the legend to Fig. 1. The scale bar is 10 microns.

Fig. 9.

UBIAD1 constitutively cycles between the ER and Golgi of isoprenoid-replete cells. A: SV-589/pMyc-UBIAD1 cells on glass coverslips of 6-well plates and cultured in medium A containing 10% FCS were injected with either 1 mg/ml BSA or purified Sar1^DN protein and 2 mg/ml Texas-Red conjugated dextran. After 3 h at 37°C, cells were fixed for immunofluorescence microscopy using IgG-9E10 (against UBIAD1) and anti-GM130. The results are representative of four independent experiments in which an average of 102 cells per experiment (408 cells total) were injected with Sar1^DN and 132 cells per experiment (528 cell total) received BSA. Approximately 92% of cells microinjected with BSA contained Golgi-localized UBIAD1 and this was reduced to 11% upon microinjection of Sar1^DN as depicted in the graph next to images. The scale bar is 10 microns. B: Schematic representation of geranylgeranyl-mediated regulation of UBIAD1 transport between the ER and Golgi.