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Fig. 4. Sterol-mediated conformational change in region of MELADL sequence of Scap. (A) mPEG-MAL labeling after sterol treatment of intact cells with cholesterol. On day 0, Insig-1- and Insig-2-deficient SRD-15 cells were set up in 60-mm dishes. On day 1, cells were transfected with 2 µg of a plasmid encoding mutant version of Scap(1–767;Cys^–) with either no cytoplasmically oriented cysteines or with a single cysteine at the indicated residue. The cells also received 0.5 µg of pCMV–Insig1–T7 as indicated. Twelve hours after transfection, cells were switched to sterol-depleting medium containing 1% HPCD for 1 h at 37°C. Cells then received sterol-depleting medium (without HPCD) with 25 µg/ml ALLN in absence or presence of 20 µM cholesterol [delivered in MCD at a cholesterol:MCD ratio (wt/wt) of 1:10]. After 3 h at 37°C, cells were harvested for preparation of sealed microsomal membrane vesicles, which were then treated with 2 mM mPEG-MAL-5000 to modify cytoplasmically exposed cysteines as described in Materials and Methods. The membranes were pelleted, solubilized, and subjected to 7% SDS/PAGE and immunoblotted with IgG-R139 (anti-Scap). (B) mPEG-MAL labeling after treatment of intact cells with 25-HC or cholesterol. On day 0, Insig-1- and Insig-2-deficient SRD-15 cells were set up in 60-mm dishes. On day 1, cells were transfected with 2.5 µg of pTK-HSV-SREBP-2 and 2 µg of pCMV–Scap(1–767;Cys^–;R445C). Cells were also transfected with 0.5 µg of pCMV–Insig1–T7 as indicated. Twelve h after transfection, cells were incubated with sterol-depleting medium containing 1% HPCD for 1 h at 37°C. Cells were then switched to sterol-depleting medium (without HPCD) with the indicated concentration of either 25-HC (dissolved in ethanol) or cholesterol (delivered in MCD). After incubation for 6 h (ALLN at 25 µg/ml added 3 h before harvest), cells were harvested, and sealed microsomal membrane vesicles were prepared and treated with 2 mM mPEG-MAL-5000 as described above. The modified membranes were subjected to 7% SDS/PAGE and immunoblot analysis with IgG-R139 (anti-Scap). (C) Trypsin cleavage of Scap after treatment of intact cells with cholesterol or 25-HC. On day 0, Scap-deficient SRD-13A cells were set up in 60-mm dishes. On day 2, cells were transfected with 2 µg of pTK-HSV-SREBP-2 and 1.25 µg of pCMV–Scap in the absence or presence of 0.6 µg pCMV-Insig-1-Myc. Twelve hours after transfection, cells were switched to sterol-depleting medium containing 1% HPCD. After incubation for 1 h at 37°C, cells were incubated with sterol-depleting medium (without HPCD) containing no sterols, 50 µM of either cholesterol or 25-HC [delivered in MCD at a sterol/MCD ratio (wt/wt) of 1:10]. After incubation for 3 h, cells were harvested, and aliquots of microsomal membranes (100 µg) were treated sequentially with 2 µg of trypsin (30°C, 30 min) and 625 units of PNGase F (37°C, 12 h) and then subjected to 12% Tris-tricine SDS/PAGE and immunoblot analysis with IgG-R139 (anti-Scap). (D and E) SREBP-2 cleavage after treatment of cells with cholesterol (D) or 25-HC (E). SRD-15 cells were set up, transfected, and treated with the indicated concentration of cholesterol (delivered in MCD) or 25-HC (dissolved in ethanol) as in B. Nuclear extract and membrane fractions were prepared and subjected to 8% SDS/PAGE and immunoblot analysis with anti-HSV IgG (anti-SREBP-2) and anti-T7 IgG (anti-Insig-1) as indicated. (F) Densitometric quantification of the bands in D and E. The intensity of the cleaved nuclear form of SREBP-2 in the absence of sterol treatment was arbitrarily set at 100%.