1. Volume 10, Issue 2, Pages 237-245 (August 2002)
Cholesterol Addition to ER Membranes Alters Conformation of SCAP, the SREBP Escort Protein that Regulates Cholesterol Metabolism 
Andrew J Brown, Liping Sun, Jamison D Feramisco, Michael S Brown, Joseph L Goldstein 
Molecular Cell 
Volume 10, Issue 2, Pages (August 2002)
DOI: /S (02)

2. Figure 1 Trypsin-Resistant Fragment of Hamster SCAP
(Top) Amino acid sequence of hamster SCAP in the region before the seventh transmembrane domain (residues 481–518) and after the eighth transmembrane domain (residues 731–773). Arginine and lysine residues (potential trypsin-cleavage sites) are denoted in blue. Arrows denote the cholesterol-dependent and independent sites of trypsin cleavage. The two palmitoylated cysteines are denoted in purple. (Bottom) Schematic model of the membrane topology of SCAP in the region of the trypsin-resistant fragment. The schematic also shows the location of the trypsin-resistant fragment that is recognized by polyclonal and monoclonal antibodies, IgG-R139 and IgG-9D5, respectively, and the two N-linked glycosylation sites (green) in the luminal loop between the seventh and eighth transmembrane domains (Nohturfft et al., 1998b).
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
Altered Trypsin Digestion of SCAP after Incubation of Intact Hamster Cells with Sterols and after Treatment of Sterol-Deprived Membranes In Vitro with Cholesterol
(A) CHO/pGFP-SCAP cells were set up for experiments and incubated for 2 hr at 37°C in the absence (− sterols) or presence (+ sterols) of 10 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as described in Experimental Procedures. The cells were harvested, and the 20,000 × g membrane suspension was treated with trypsin at 30°C for the indicated time, followed by treatment with PNGase F. The proteins were then subjected to SDS-PAGE on 16% gels and immunoblot analysis with anti-SCAP IgG-9D5. The filter was exposed to film for 2 min.
(B) CHO/pGFP-SCAP cells were set up for experiments and incubated with sterol-depleting medium as described in Experimental Procedures. Aliquots of resuspended membranes were incubated with cholesterol complexed with MCD under standard conditions: room temperature, cholesterol concentration of 25 μM, and incubation for 20 min as described in Experimental Procedures. Times of incubation, temperatures of incubation, and concentrations of cholesterol were varied as indicated. At the end of the incubation, the membranes were treated sequentially with trypsin (20 min at 30°C) followed by PNGase F, and then subjected to 16% SDS-PAGE and immunoblot analysis with anti-SCAP IgG-9D5. The filters were exposed to film for 15–30 s.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Sterol-Resistant SCAP Mutants Are Insensitive to Trypsin Digestion after Treatment of Membranes In Vitro with Cholesterol
(A) On day 0, wild-type CHO/pSCAP cells and sterol-resistant mutant CHO/pSCAP(Y298C) and CHO/pSCAP(D443N) cells were set up for experiments in medium C supplemented with 5% fetal calf lipoprotein-deficient serum at the following densities per 100 mm dish to give comparable cell densities at the time of harvest: 8 × 105, 10 × 105, and 6 × 105 cells, respectively. On day 2, the cells were switched to medium C supplemented with 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, and 50 μM sodium mevalonate. After incubation for 16 hr at 37°C, the cells were harvested. Aliquots of the 20,000 × g membrane suspension were incubated at room temperature for 20 min with cholesterol/MCD at a cholesterol concentration of 5 μM. At the end of the incubation, the membranes were treated sequentially with trypsin (20 min at 30°C) and PNGase F and then subjected to 16% SDS-PAGE and immunoblot analysis with anti-SCAP IgG-R139. The filter was exposed to film for 30 s.
(B) SRD-13A cells were transiently transfected with wild-type or the Y298C version of pCMV-SCAP as described in Experimental Procedures. After the cells were harvested, the 20,000 × g membrane suspension was incubated for 20 min at room temperature with the indicated concentration of cholesterol complexed to MDC, treated sequentially with trypsin (30 min at 30°C) and PNGase F, and processed for immunoblot analysis as described above. The filter was exposed to film for 2 s. Relative intensity of the upper and lower bands was quantified by densitometry.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Specificity of Exogenously Added Sterols in Altering Trypsin Digestion of SCAP
CHO/pGFP-SCAP cells were set up for experiments, incubated in sterol-depleting medium, and harvested as described in Experimental Procedures. The 20,000 × g membrane suspension was incubated with the indicated sterol/MCD complex at a sterol concentration of 25 μM for 20 min at room temperature, treated sequentially with trypsin (20 min at 30°C) and PNGase F, and then processed for immunoblot analysis as described in Figure 3. The data in (A) and (B) are from two independent experiments. The filters were exposed to film for 30 s (A) and 45 s (B).
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Mapping the COOH-Terminal Cleavage Site in Trypsin-Resistant Fragment of SCAP
SRD-13A cells were transiently transfected with wild-type or mutant versions of pCMV-SCAP as described in Experimental Procedures. After the cells were harvested, the 20,000 × g membrane suspension was incubated in the absence (−) or presence (+) of cholesterol/MCD at a cholesterol concentration of 50 μM for 20 min at room temperature, treated sequentially with trypsin (30 min at 30°C) and PNGase F, and processed for immunoblot analysis anti-SCAP IgG-R139. The filter was exposed to film for 3 s.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Palmitoylation of Transfected SCAP and Its Lack of Effect on SREBP-2 Cleavage in Hamster Cells
(A) Trypsin-resistant fragment of palmitoylated SCAP. HEK-293 cells were transfected with pCMV-SCAP, incubated with [3H]palmitate, and harvested as described in Experimental Procedures. A suspension of the 20,000 × g membrane fraction was treated with trypsin for 30 min at 30°C, followed by incubation in the absence or presence of PNGase F, and then immunoprecipitated with anti-SCAP IgG-139 as described in Experimental Procedures. The samples were subjected to 15% SDS-PAGE and transferred to nitrocellulose for immunoblotting or incubated with fluorographic reagent and dried for fluorography. The filter was blotted with anti-SCAP IgG-9D5 and exposed to film for 90 s (immunoblot). The dried gel for fluorography was exposed to preflashed film for 3 days (fluorogram).
(B) Location of palmitoylated cysteines in hamster SCAP as determined by site-directed mutagenesis. HEK-293 cells were transfected with vector alone (lane 1, mock), wild-type pCMV-SCAP (lane 2), or mutant versions of pCMV-SCAP (lanes 3–6) and metabolically labeled with [3H]palmitate as described in Experimental Procedures. The cells were lysed in RIPA buffer, aliquots were immunoprecipitated with anti-SCAP IgG-R139, and the immunoprecipitates were subjected to 8% SDS-PAGE and transferred to nitrocellulose for immunoblotting or incubated with fluorographic reagent and dried for fluorography. The filter was blotted with anti-SCAP IgG-9D5 and exposed to film for 1 s (immunoblot). The dried gel for fluorography was exposed to preflashed film for 16 hr (fluorogram). The intensity of bands on the fluorogram from three independent experiments (determined by densitometry) was averaged, and the wild-type value (lane 2) was assigned a relative intensity of 1.0.
(C) Palmitoylation is not required for SCAP function. On day 0, wild-type CHO/pSCAP cells and the nonpalmitoylated mutant CHO/pSCAP(C728S,C734S) cells were set up for experiments as described in Experimental Procedures. On day 3, cells were incubated for 5 hr at 37°C in medium C supplemented with 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and the indicated concentrations of 25-hydroxycholesterol (25-HC) plus 10 μg/ml of cholesterol and then harvested for preparation of cell fractions. Aliquots of membranes (20 μg) and nuclear extracts (35 μg) were subjected to 8% SDS-PAGE and immunoblot analysis with IgG-7D4 for SREBP-2 (membranes and nuclear extracts) and IgG-R139 for SCAP (membranes only). P and N denote the precursor and cleaved nuclear forms of SREBP-2, respectively. Filters were exposed to film for 4 s (SREBP-2, membranes), 30 s (SREBP-2, nuclear extracts), and 3 s (SCAP).
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7 Model for Cholesterol-Induced Conformational Change in SCAP
The addition of cholesterol to membranes changes the conformation of SCAP so as to render residues proximal to the seventh transmembrane domain more accessible to trypsin. In membranes from sterol-deprived cells, trypsin cleaves SCAP on its NH2-terminal side at R496 (top). Incubation of the same membranes with cholesterol in vitro creates a new trypsin cleavage site on the NH2-terminal side at R503 and R505 (bottom). The trypsin cleavage site on the COOH-terminal side of SCAP occurs within the same cluster of arginines (R ) in the absence and presence of cholesterol. Two point mutations, Y298C and D443N, render the sterol-sensing domain of SCAP resistant to the cholesterol-induced conformational change; their location is denoted by the green boxes (top).
Molecular Cell  , DOI: ( /S (02) )