Figure 8. 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% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1. (B) SV-589 cells were set up on day 0 at 3 × 10⁴ cells/well of a twelve-well plate with a glass coverslip in medium A containing 10% FCS. On day 1, the cells were transfected using FuGENE 6 with 50 ng of wild type (WT), N102S, or G177R versions of pCMV-Myc-UBIAD1; the total amount of DNA/well was adjusted to 500 ng by the addition of pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS (final concentration). After 16 hr at 37°C, cells were fixed and analyzed by microscopy as described in ‘Materials and methods’.

DOI: http://dx.doi.org/10.7554/eLife.05560.016

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 day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1.

DOI: http://dx.doi.org/10.7554/eLife.05560.017

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 described in the legend to Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.05560.018