Figure 1.

Expression of HMG CoA reductase protein is reduced in absence of UBIAD1 and enhanced in presence of SCD-associated UBIAD1 (N102S). A and B, SV-589, SV-589(ΔUBIAD1), SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT), and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 4 × 10⁵ cells/60-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were refed medium A supplemented with either 10% FCS or LPDS as indicated. After 16 h at 37 °C, the cells were harvested for subcellular fractionation (A) or preparation of total RNA (B) as described under “Experimental procedures.” A, aliquots of membrane fractions (20 μg protein/lane) were subjected to SDS-PAGE, and immunoblot analysis was carried with IgG-A9 (against HMG CoA reductase), IgG-H8 (against endogenous UBIAD1), IgG-D1 (against ACAT-1), and anti-calnexin IgG. B, total RNA was subjected to quantitative RT-PCR using primers against human reductase; the human 36B4 mRNA was used as an invariant control. Each value represents the amount of reductase mRNA relative to that in SV-589 cells, which is arbitrarily defined as 1. C, SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 3 × 10⁵ cells/60-mm dish in medium A containing 10% FCS. On day 1, the cells were refed identical medium and incubated for 16 h at 37 °C. The cells were then fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) using a Zeiss Axio Observer Epifluorescence microscope as described under “Experimental procedures.” D, SV-589, SV-589(ΔUBIAD1), SV-589(ΔUBIAD1)/pMyc-UBIAD1 (WT), and SV-589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 5 × 10⁵ cells/100-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were depleted of sterols through incubation in medium A supplemented with 10% LPDS, 10 μm compactin, and 50 μm mevalonate. After 16 h at 37 °C, the cells were refed the identical medium containing 10 μm MG-132, and the indicated concentration of 25-HC. Following incubation for 1 h at 37 °C, the cells were harvested, lysed in detergent-containing buffer, and immunoprecipitated with 30 μg of polyclonal IgG-839 (against reductase) as described under “Experimental procedures.” Aliquots of the resulting immunoprecipitates were then subjected to SDS-PAGE, followed by immunoblot analysis with IgG-A9 (against reductase) and IgG-P4D1 (against ubiquitin). The results in this and other figures are representative of at least two independent experiments. MW, molecular mass.