Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase - PubMed (original) (raw)

Organization of complex between HMG-CoA reductase, Insig-1, gp78, and SPFH2 as determined by co-immunoprecipitation. A, HEK-293 cells were set for experiments on day 0, depleted of sterols on day 2, and treated in the absence or presence of 10 μ

MG-132 and 1 μg/ml 25-HC plus 10 m

mevalonate (Mev.) on day 3 as described in the legend to Fig. 1_B_. After treatments the cells were harvested, lysed, and subjected to immunoprecipitation (Immunoppt.) with polyclonal antibodies against reductase (Red.). Aliquots of the resulting pellet and supernatant fractions of the immunoprecipitation were subjected to SDS-PAGE, and immunoblot analysis was carried out with monoclonal IgG-A9 (against reductase), polyclonal anti-gp78, anti-SPFH2, and monoclonal anti-VCP/p97 IgGs. Ab, antibody; Preim., Preimmune. B, CHO-7 cells were set up for experiments on day 0, transfected with 1 μg of pCMV-HMG-Red-T7, 0.1 μg of pCMV-Insig-1-Myc, and 0.1 μg of pCMV-SPFH2-Myc, and depleted of sterols on day 1 as described in the legend to Fig. 2. The sterol-depleted cells were then incubated for 2 h at 37 °C in medium A containing 5% LPDS, 10 μ

compactin, and 10 μ

MG-132 in the absence or presence of 1 μg/ml 25-HC plus 10 m

mevalonate as indicated. The cells were subsequently harvested, lysed, and subjected to anti-T7 immunoprecipitation. Aliquots of the resulting supernatant and pellet fractions were subjected to SDS-PAGE followed by immunoblot analysis with polyclonal anti-T7 IgG (against reductase) and monoclonal IgG-9E10 (against Insig-1 and SPFH2). C, CHO-7 cells were set up on day 0, transfected with 0.1 μg of pCMV-Insig-1-T7, 0.01 μg of pCMV-gp78-Myc, and 0.1 μg of pCMV-SPFH2-Myc and depleted of sterols on day 1 as described in B. After sterol depletion, the cells were incubated for 2 h at 37 °C in medium A supplemented with 5% LPDS, 10 μ

compactin, and 10 μ

MG-132. The cells were subsequently harvested and lysed, and immunoprecipitation was carried out with anti-T7-coupled beads. Aliquots of the pellet and supernatant fractions of the immunoprecipitation were subjected to SDS-PAGE and immunoblot analysis with polyclonal anti-T7 IgG (against Insig-1) and monoclonal IgG-9E10 (against gp78 and SPFH2). D, CHO-7 cells were set up on day 0, transfected with 0.1 μg/dish pCMV-SPFH2-T7 together with 0.1 μg/dish pCMV-gp78-Myc (lanes 1 and 2, WT), (lanes 3 and 4, TM), or (lanes 5 and 6, Cyto.) as indicated and depleted of sterols on day 1 as described in A. After sterol depletion, the cells were subjected to incubation for 2 h in medium A containing 5% LPDS and 10 μ

compactin with or without 1 μg/ml 25-HC plus 10 m

mevalonate as indicated. The cells were then harvested, lysed, and immunoprecipitated with anti-T7 beads. Aliquots of the pellet and supernatant fractions were subjected to SDS-PAGE followed by immunoblot analysis with polyclonal anti-T7 (against SPFH2) and monoclonal IgG-9E10 (against gp78).