1. Volume 85, Issue 7, Pages 1037-1046 (June 1996)
Sterol-Regulated Release of SREBP-2 from Cell Membranes Requires Two Sequential Cleavages, One Within a Transmembrane Segment 
Juro Sakai, Elizabeth A Duncan, Robert B Rawson, Xianxin Hua, Michael S Brown, Joseph L Goldstein 
Cell 
Volume 85, Issue 7, Pages (June 1996)
DOI: /S (00)

2. Figure 1
Model for the Two-Step Proteolytic Cleavage of Membrane-Bound SREBPs
(A) The first cleavage of the membrane-bound precursor (P form) occurs in the lumen of the ER (denoted by the bold arrow) when cells are depleted of sterols. This reaction requires the presence of an arginine in the lumenal loop (denoted by R).
(B) The resulting NH2-terminal intermediate (I form) remains membrane-bound until it is cleaved within the first transmembrane domain by a protease that is not regulated by sterols (denoted by the bold arrow). This reaction requires the DRSR sequence on the NH2-terminal side of the first transmembrane segment.
(C) After the second cleavage, the mature NH2-terminal portion of SREBP (M form) is released from the membrane and travels to the nucleus, where it activates transcription. Although this figure depicts the SREBP as a monomer, it is likely that it exists as a dimer formed by interactions between the basic–helix-loop-helix–leucine zipper domains.
Cell  , DOI: ( /S (00) )

3. Figure 2
Immunoblot Analysis of 293 Cells Transfected with Mutant Forms of Epitope-Tagged SREBP-2
On day 0, 293 cells were set up at 4 × 105 cells per 60 mm dish in medium B (Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin) supplemented with 10% fetal calf serum. On day 2, cells were transfected with 5 μg/dish of pTK–HSV–BP-2 as previously described (Hua et al. 1995). Mock-transfected cells (lanes 1 and 2) received 5 μg/dish of empty vector pTK (Hua et al. 1995). After transfection (3 hr), the cells were changed to medium B containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, and 50 μM mevalonate in the absence (minus) or presence (plus) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol (sterols) as indicated. All cultures were adjusted to contain 0.2% ethanol. On day 3, 4 hr before the harvest, all cells received 25 μg/ml N-acetyl-leucine-leucine-norleucinal. The cells were harvested, and the nuclear extract and membrane fractions were prepared as described in Experimental Procedures. Aliquots of membranes (80 μg) and nuclear extract (60 μg) were subjected to SDS–PAGE and immunoblot analysis with 0.5 μg/ml HSV-TagTM antibody. P, I, and M denote the precursor, intermediate, and mature forms of SREBP-2, respectively. The filters were exposed to film for 15 s.
Cell  , DOI: ( /S (00) )

4. Figure 5
Extraction of Precursor and Membrane-Bound Intermediate Forms of H-Ras–SREBP-2 Fusion Protein in Transfected 293 Cells
Cells were transfected with 7.5 μg/dish of plasmid B (see Figure 3A) and incubated for 24 hr in medium B containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, and 50 μM mevalonate without sterols. The 105 g membrane fraction from two 60 mm dishes of cells (360 μg protein) was resuspended in 0.3 ml of the following solutions: lane 1, buffer B; lane 2, 0.1 M Na2CO3 (pH 11); lane 3, 1% (v/v) Triton X-100 in buffer B. After incubation for 30 min at 4°C in a rotating apparatus, each mixture was centrifuged at 105 g for 45 min at 4°C in a Beckman TLA rotor. Each pellet was resuspended in 0.3 ml of SDS lysis buffer. Equal aliquots (0.14 ml) of the resuspended pellet (lanes 4–6) and the 105 g supernatant (lanes 1–3) were subjected to SDS–PAGE and immunoblotted with 1 μg/ml HSV-TagTM antibody. The filters were exposed to film for 5 s. Ras P and Ras I denote the precursor and intermediate forms of Ras–SREBP-2 fusion proteins, respectively. Figure 3 Immunoblot Analysis of 293 Cells Transfected with Epitope-Tagged H-Ras–SREBP-2 (A) Domain structures of human H-Ras–SREBP-2 fusion proteins, indicating the region replaced with human H-Ras and the site of the D478RSR→AS mutation. The expression vector was constructed as described in Experimental Procedures. Chimeric plasmid A, designated pTK–HSV– Ras–BP2 (473–1141), was constructed by replacing the NH2-terminal region of pTK–HSV–BP2 (residues 14–472) with human H-Ras (residues 2–189). Chimeric plasmid B, designated pTK–HSV–Ras–BP2 (473–1141, D478RSR→AS), was constructed by replacing the 4 amino acid sequence DRSR (residues 478–481) with AS, as described in Experimental Procedures. (B) Immunoblot analysis of membrane and cytosol fractions from 293 cells transfected with 3 μg/dish of the indicated HSV tagged human H-Ras–SREBP-2 plasmid. Cells were set up for experiments, transfected with indicated plasmid, incubated in the absence (minus) or presence (plus) of 3 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol, as indicated. Membrane and cytosol fractions were prepared as described in Experimental Procedures. Aliquots of 100 μg of membrane protein (upper panel) and 120 μg of cytosol protein (lower panel) were subjected to SDS–PAGE and immunoblotted with 0.5 μg/ml HSV-TagTM antibody. The filters were exposed to film for 30 s and 15 s for the membrane and cytosol fractions, respectively. Ras P, Ras I, and Ras M denote the precursor, intermediate, and mature forms of Ras–SREBP-2 fusion protein, respectively. The asterisk denotes a weak band of unknown significance in the cells transfected with plasmid B. (C) Aliquots (100 μg protein) of the cytosol fraction (designated C) from lane 3 and the membrane fraction (designated M) from lane 5 in (B) were subjected to SDS–PAGE in adjacent lanes, followed by immunoblot analysis. Ras I and Ras M denote the intermediate and mature forms of Ras–SREBP-2 fusion protein, respectively. The filters were exposed to film for 2 min.
Cell  , DOI: ( /S (00) )

5. Figure 5
Extraction of Precursor and Membrane-Bound Intermediate Forms of H-Ras–SREBP-2 Fusion Protein in Transfected 293 Cells
Cells were transfected with 7.5 μg/dish of plasmid B (see Figure 3A) and incubated for 24 hr in medium B containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, and 50 μM mevalonate without sterols. The 105 g membrane fraction from two 60 mm dishes of cells (360 μg protein) was resuspended in 0.3 ml of the following solutions: lane 1, buffer B; lane 2, 0.1 M Na2CO3 (pH 11); lane 3, 1% (v/v) Triton X-100 in buffer B. After incubation for 30 min at 4°C in a rotating apparatus, each mixture was centrifuged at 105 g for 45 min at 4°C in a Beckman TLA rotor. Each pellet was resuspended in 0.3 ml of SDS lysis buffer. Equal aliquots (0.14 ml) of the resuspended pellet (lanes 4–6) and the 105 g supernatant (lanes 1–3) were subjected to SDS–PAGE and immunoblotted with 1 μg/ml HSV-TagTM antibody. The filters were exposed to film for 5 s. Ras P and Ras I denote the precursor and intermediate forms of Ras–SREBP-2 fusion proteins, respectively. Figure 3 Immunoblot Analysis of 293 Cells Transfected with Epitope-Tagged H-Ras–SREBP-2 (A) Domain structures of human H-Ras–SREBP-2 fusion proteins, indicating the region replaced with human H-Ras and the site of the D478RSR→AS mutation. The expression vector was constructed as described in Experimental Procedures. Chimeric plasmid A, designated pTK–HSV– Ras–BP2 (473–1141), was constructed by replacing the NH2-terminal region of pTK–HSV–BP2 (residues 14–472) with human H-Ras (residues 2–189). Chimeric plasmid B, designated pTK–HSV–Ras–BP2 (473–1141, D478RSR→AS), was constructed by replacing the 4 amino acid sequence DRSR (residues 478–481) with AS, as described in Experimental Procedures. (B) Immunoblot analysis of membrane and cytosol fractions from 293 cells transfected with 3 μg/dish of the indicated HSV tagged human H-Ras–SREBP-2 plasmid. Cells were set up for experiments, transfected with indicated plasmid, incubated in the absence (minus) or presence (plus) of 3 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol, as indicated. Membrane and cytosol fractions were prepared as described in Experimental Procedures. Aliquots of 100 μg of membrane protein (upper panel) and 120 μg of cytosol protein (lower panel) were subjected to SDS–PAGE and immunoblotted with 0.5 μg/ml HSV-TagTM antibody. The filters were exposed to film for 30 s and 15 s for the membrane and cytosol fractions, respectively. Ras P, Ras I, and Ras M denote the precursor, intermediate, and mature forms of Ras–SREBP-2 fusion protein, respectively. The asterisk denotes a weak band of unknown significance in the cells transfected with plasmid B. (C) Aliquots (100 μg protein) of the cytosol fraction (designated C) from lane 3 and the membrane fraction (designated M) from lane 5 in (B) were subjected to SDS–PAGE in adjacent lanes, followed by immunoblot analysis. Ras I and Ras M denote the intermediate and mature forms of Ras–SREBP-2 fusion protein, respectively. The filters were exposed to film for 2 min.
Cell  , DOI: ( /S (00) )

6. Figure 3
Immunoblot Analysis of 293 Cells Transfected with Epitope-Tagged H-Ras–SREBP-2
(A) Domain structures of human H-Ras–SREBP-2 fusion proteins, indicating the region replaced with human H-Ras and the site of the D478RSR→AS mutation. The expression vector was constructed as described in Experimental Procedures. Chimeric plasmid A, designated pTK–HSV– Ras–BP2 (473–1141), was constructed by replacing the NH2-terminal region of pTK–HSV–BP2 (residues 14–472) with human H-Ras (residues 2–189). Chimeric plasmid B, designated pTK–HSV–Ras–BP2 (473–1141, D478RSR→AS), was constructed by replacing the 4 amino acid sequence DRSR (residues 478–481) with AS, as described in Experimental Procedures.
(B) Immunoblot analysis of membrane and cytosol fractions from 293 cells transfected with 3 μg/dish of the indicated HSV tagged human H-Ras–SREBP-2 plasmid. Cells were set up for experiments, transfected with indicated plasmid, incubated in the absence (minus) or presence (plus) of 3 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol, as indicated. Membrane and cytosol fractions were prepared as described in Experimental Procedures. Aliquots of 100 μg of membrane protein (upper panel) and 120 μg of cytosol protein (lower panel) were subjected to SDS–PAGE and immunoblotted with 0.5 μg/ml HSV-TagTM antibody. The filters were exposed to film for 30 s and 15 s for the membrane and cytosol fractions, respectively. Ras P, Ras I, and Ras M denote the precursor, intermediate, and mature forms of Ras–SREBP-2 fusion protein, respectively. The asterisk denotes a weak band of unknown significance in the cells transfected with plasmid B.
(C) Aliquots (100 μg protein) of the cytosol fraction (designated C) from lane 3 and the membrane fraction (designated M) from lane 5 in (B) were subjected to SDS–PAGE in adjacent lanes, followed by immunoblot analysis. Ras I and Ras M denote the intermediate and mature forms of Ras–SREBP-2 fusion protein, respectively. The filters were exposed to film for 2 min.
Cell  , DOI: ( /S (00) )

7. Figure 6
Immunoblot Analysis of 293 Cells Transfected with Truncated Forms of Epitope-Tagged H-Ras–SREBP-2
A schematic diagram depicting the location of the stop mutations in the HSV tagged H-Ras–SREBP-2 fusion protein is shown at the top.
(A) Cells were transfected with 5 μg/dish of wild-type or the indicated mutant pTK–HSV–Ras–BP2 expression vector and incubated for 24 hr in medium B containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, and 50 μM sodium mevalonate in the absence of sterols, as described in the legend to Figure 2. Aliquots of the membrane (150 μg protein for lanes 1, 2, and 4–7 and 5 μg for lane 3) and cytosol (200 μg protein for lanes 1, 2, and 7 and 3–7 μg for lanes 3–6) fractions were subjected to SDS–PAGE and immunoblot analysis with 1 μg/ml HSV-TagTM antibody. The filters were exposed to film for 30s and 15 s for the membrane and cytosol fractions, respectively.
(B) Aliquots of the cytosol fraction (designated C; 10 μg protein) and membrane fraction (designated M; 69 μg protein) from the Stop 503 transfected cells in lane 4 were subjected to SDS–PAGE in adjacent lanes, followed by immunoblot analysis. The filters were exposed to film for 45 s.
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8. Figure 4
Estimation of the Size of the Intermediate Form Generated from the DRSR→AS Mutant of H-Ras–SREBP-2 Fusion Protein
293 cells were transfected with 3 μg/dish of the indicated mutant form of HSV tagged H-Ras–SREBP-2 plasmid, as described in the legend to Figure 3. Aliquots of the membrane fraction (42 μg protein in lanes 1 and 2 and 138 μg protein in lane 3) were subjected to SDS–PAGE in adjacent lanes, followed by immunoblot analysis. The filters were exposed to film for 30 s. Ras I denotes the intermediate form of the DRSR→AS mutant protein. The asterisk denotes the mature form of Ras–SREBP-2 fusion protein that was found in the membrane fraction.
Cell  , DOI: ( /S (00) )

9. Figure 7
Immunoblot Analysis of 293 Cells Transfected with the Double Mutant Stop 519–D478RSR→AS Version of Epitope-Tagged SREBP-2
Cells were set up and transfected with 5 μg/dish of the indicated wild-type or mutant form of pTK–HSV–BP2, as described in the legend to Figure 2. Aliquots of membranes (80 μg protein) and nuclear extract (60 μg protein) were subjected to SDS–PAGE and immunoblot analysis with 0.5 μg/ml HSV-TagTM antibody. The filters were exposed to film for 7 s.
Cell  , DOI: ( /S (00) )

10. Figure 8
Immunoblot Analysis of Endogenous SREBP-1 and SREBP-2 in Wild-Type CHO Cells and Mutant CHO Cell Lines That Are Auxotrophic for Cholesterol (SRD-6B and M19 Cells)
On day 0, the indicated cells were set up as described in Experimental Procedures. On day 2, the medium was changed to medium A containing 5% fetal calf lipoprotein-deficient serum and 50 μM sodium mevalonate supplemented with either 50 μM compactin (plus compactin) or 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol (plus sterols), as indicated. On day 3, 4 hr before the harvest, all cells received 25 μg/ml N-acetyl-leucine-leucine-norleucinal. The cells were harvested, fractionated as described in Experimental Procedures, and aliquots (50 μg protein) of the membrane and nuclear extract fractions were subjected to SDS–PAGE. Immunoblot analysis was carried out with 15 μg/ml IgG-2A4 for SREBP-1 (A) or 7.5 μg/ml IgG-7D4 antibody for SREBP-2 (B). In (A), filters for both fractions were exposed to film for 60 s; in (B), filters for the membrane and nuclear extracts fractions were exposed to film for 2 min and 15 s, respectively. P, I, and M denote the precursor, intermediate, and mature forms of SREBP-1 or SREBP-2, respectively. The asterisk in the membrane fraction of CHO-7 cells (lane 11) denotes a 62 kDa band that migrates faster than the intermediate (I) form of SREBP-2 in lanes 13, 14, 16, and 17. This 62 kDa band in lane 11 is believed to represent contamination with nuclei containing the mature form (M) of SREBP-2.
Cell  , DOI: ( /S (00) )