The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase
- University of Texas Southwestern Medical Center, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Biosynthesis of cholesterol and menaquinone-4 (MK-4, vitamin K2) in mammalian cells.
https://doi.org/10.7554/eLife.05560.003
Figure 2
Insig-mediated, sterol-accelerated degradation of HMG CoA reductase in mammalian cells.
https://doi.org/10.7554/eLife.05560.004
Figure 3 with 1 supplement
Identification of UBIAD1 as an associated protein of HMG CoA reductase.
(A) Amino acid sequence and predicted topology of human UBIAD1. Amino acid residues mutated in Schnyder corneal dystrophy (SCD) families are shaded in red; asparagine-102 (N102) and glycine-177 …
Figure 3—figure supplement 1
Identification of proteins associated with HMG-Red(TM1-8)-BirA*.
(A) HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0, depleted of sterols on day 3, harvested on day 4 for lysis and affinity purification using streptavidin-coupled beads as described in …
Figure 4
Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.
SV-589 cells were set up on day 0 at 1 × 105 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, …
Figure 5 with 3 supplements
The nonsterol isoprenoid geranylgeraniol inhibits sterol-induced binding of UBIAD1 to HMG CoA reductase and promotes its translocation to the Golgi.
(A and B) SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing …
Figure 5—figure supplement 1
The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase.
SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing 10% NC-LPPS, …
Figure 5—figure supplement 2
Geranylgeraniol, but not 25-HC, FOH, or cholesterol, stimulates translocation of endogenous UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.
(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium …
Figure 5—figure supplement 3
Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.
SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium …
Figure 6 with 1 supplement
RNA interference-mediated knockdown or CRISPR/Cas9-mediated knockout of UBIAD1 alleviates geranylgeraniol requirement in sterol-accelerated degradation of HMG CoA reductase.
(A and B) SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 4. The …
Figure 6—figure supplement 1
RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation.
SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3. Notably, the siRNA duplex …
Figure 7 with 1 supplement
The Schnyder corneal dystrophy (SCD)-associated N102S and G177R mutants of UBIAD1 block sterol-accelerated ERAD of HMG CoA reductase.
SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red(TM1-8)-T7 in the …
Figure 7—figure supplement 1
Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase.
SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or …
Figure 8 with 2 supplements
SCD-associated UBIAD1 mutant resists geranylgeraniol-mediated displacement from HMG CoA reductase and remains sequestered in ER membranes.
(A) UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% …
Figure 8—figure supplement 1
SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments (A and B).
UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On …
Figure 8—figure supplement 2
Subcellular localization of wild type and SCD-associated N102S UBIAD1 in transfected SV-589 cells.
SV-589 cells were set up for experiments on day 0, transfected on day 1 with pCMV-Myc-UBIAD1 (WT) or (N102S) in medium A containing 10% FCS, and subjected to immunostaining, followed by imaging as …
Figure 9
Proposed model for role of UBIAD1 in sterol-accelerated degradation of HMG CoA reductase.
In sterol-deprived cells, both reductase and UBIAD1 localize to membranes of the ER. The intracellular accumulation of sterols in ER membranes triggers binding of reductase to Insigs, resulting in …
