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. 2021 Nov 29;10:e64688. doi: 10.7554/eLife.64688

Figure 1. Overexpression of PDP1 inhibits protein geranylgeranylation and GGOH-enhanced ERAD of HMG CoA reductase.

(A) SV-589/PDP1-Myc-FLAG cells were set up on day 0 at 2 × 105 cells per 60 mm dish in medium A supplemented with 5 % FCS. On day 1, cells were refed identical medium in the absence or presence of tetracycline (1 µg/ml) and the indicated amount of azido-geranylgeraniol (azido-GGOH). Following incubation for 16 hr at 37 °C, cells were harvested, lysed and proteins were precipitated. The resulting material was then resuspended in buffer, labeled with biotin alkyne, and subjected to SDS-PAGE followed by streptavidin blotting as described in ‘Materials and methods’. (B and C) SV-589/PDP1-Myc-FLAG cells were set up on day 0 at 3 × 105 cells per 100 mm dish in medium A supplemented with 5 % FCS. (B) On day 3, cells were refed the identical medium containing 10 µM sodium compactin and 50 µM sodium mevalonate in the absence or presence of tetracycline (1 µg/ml). Following incubation for 16 hr at 37 °C, cells were treated with the indicated amount of GGOH and incubated an additional 6 hrs at 37 °C. Cells were then harvested, lysed, and subjected to subcellular fractionation. Aliquots of resulting membrane fractions (50 µg protein loaded/lane) were subjected to SDS-PAGE, followed by immunoblot analysis with IgG-A9 (against reductase), anti-PDP1 IgG, IgG-1H12 (against UBIAD1), and anti-calnexin IgG. (C) On day 3, cells were depleted of sterol and nonsterol isoprenoids through incubation in medium A containing 10 % LPDS, 10 µM compactin, and 50 µM mevalonate; some of the cells also received 1 µg/ml tetracycline as indicated. After 16 hr at 37 °C, the cells were treated with or without 1 µg/ml 25-HC and the indicated amount of GGOH. Following incubation for 3 hr at 37 °C, cells were harvested, lysed, and subjected to subcellular fractionation. Aliquots of resulting membrane and nuclear extract fractions (30–50 µg protein loaded/lane) were analyzed by immunoblot using IgG-A9 (against reductase), anti-PDP1 IgG, IgG-1H12 (against UBIAD1), anti-calnexin IgG, IgG-22D5 (against SREBP-2), and anti-LSD-1 IgG.

Figure 1.

Figure 1—figure supplement 1. Overexpression of catalytically inactive PDP1 (S212T) enhances ERAD of HMG CoA reductase.

Figure 1—figure supplement 1.

SV-589/TR cells were set up on day 0 at 2.4 × 105 cells per 60 mm dish in medium A supplemented with 5 % FCS. On day 1, cells were transfected with 1 µg/dish pCMV-HMGCR (TM1-8)-T7 in the absence or presence of 0.25 µg/dish pCMV-PDP1 (S212T)-FLAG using FuGENE6 as described in ‘Materials and Methods’. Following incubation for 16 hr at 37 °C, cells were treated in the absence or presence of 30 µM GGOH for 4 hr after which they were harvested, lysed and subjected to subcellular fractionation. Aliquots of resulting membrane fractions (50 µg protein loaded/lane) were analyzed by immunoblot using anti-T7•Tag monoclonal antibody (against reductase), anti-FLAG M2 (against PDP1 (S212T), and anti-calnexin IgG).