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Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins

Li-Ping Sun^*, Joachim Seemann, Joseph L. Goldstein^*,, and Michael S. Brown^*,

Departments of *Molecular Genetics and Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390

View larger version (38K): [in this window] [in a new window]   Fig. 1. Amino acid sequence and topology of the membrane domain of hamster Scap. The cytoplasmic loop between transmembrane helices 6 and 7 is referred to as loop 6, and it is postulated to extend from Ser-441 to Arg-518. Amino acids 447–452 (highlighted in red) constitute the hexapeptide sequence MELADL. Filled arrows denote amino acids that were mutated in the current experiments. Open arrows denote all of the cysteine residues in the membrane domains of Scap; those cysteines marked by asterisks (*) were changed to alanines to create the Cys(–) construct used as the parent plasmid for the experiments in Fig. 4. Arg-505 (highlighted in yellow) denotes a cholesterol-induced, trypsin-sensitive cleavage site (20, 21). Trypsin-resistant fragment denotes the sequence between membrane helices 7 and 8 (amino acids 540–707) that is detected by IgG-R139 (33).

View larger version (50K): [in this window] [in a new window]   Fig. 2. Anti-MELADL inhibits Scap transport from ER to Golgi. (A) Binding of COPII proteins to Scap in vitro. On day 0, Scap-deficient SRD-13A cells were set up in 100-mm dishes. On day 2, cells were transfected with 2 µg of pTK-SREBP-2, 0.4 µg of pCMV–Scap, and 0.2 µg of pCMV-Insig-1-Myc. Twelve hours later, cells were switched to sterol-depleting medium containing 1% HPCD and incubated for 1 h at 37°C. Cells were then switched to the same medium without HPCD. After 3 h at 37°C, cells were harvested, and 150 µg of microsomal membranes (16,000 x g pellet) were incubated, in a final volume of 0.3 ml Buffer B, with the indicated amount of affinity-purified control anti-T7 tag or anti-MELADL antibody in absence or presence of increasing amounts (0.2, 0.5, and 1.0 mg) of a 16-aa synthetic peptide corresponding to residues 446–461 of Scap and containing wild-type (lanes 6–8) or a mutant MELADL sequence substituted with AAAAAA (lanes 9–11). After a 30-min incubation on ice, we added 10 µg of a recombinant mutant of GST-Sar-1(H79G; GTPase-defective) and 10 µg of recombinant Flag-Sec23/24 in the presence of 0.5 mM sodium GTP. The Scap/COPII complex was precipitated with anti-FLAG. The resulting supernatant (Sup.) and pellet (5% of Sup.) fractions were subjected to 8% SDS/PAGE and immunoblot analysis with IgG-R139 (anti-Scap). (B) Immunofluorescence. On day 0, CHO/pGFP-Scap cells were set up on 12-mm coverslips as described in Materials and Methods. On day 2, cells were switched to microinjection medium supplemented with 5% FCS, after which Fab fragments (0.2 mg/ml) of either anti-MELADL or control antibody (IgG-R139) were microinjected into the cytoplasm. After 1-h incubation at 37°C, cells were switched to sterol-depleting medium containing 1% HPCD without sterols and incubated for 1 h at 37°C. Cells were then fixed for 15 min in 3.7% formaldehyde in PBS at room temperature and permeabilized for 10 min in methanol at –20°C. Cells were stained with monoclonal IgG-GM130 (1.2 µg/ml, anti-GM130) and visualized with Alexa Fluor 594 goat anti-mouse IgG (1:300 dilution, GM130), and Alexa Fluor 350 goat anti-rabbit IgG (1:200 dilution, microinjected Fab fragments). GFP-Scap was visualized directly. (Scale bar, 25 µm.) (C) Time-lapse imaging. On day 0, CHO/pGFP-Scap cells were set up on 18-mm coverslips as described in Materials and Methods. On day 1, cells were switched to medium A with 5% LPDS and 1% HPCD in the presence of sterols (10 µg/ml 25-HC and 10 µg/ml cholesterol in 0.2% ethanol) and incubated for 16 h. On day 2, cells were refed with microinjection medium with 5% LPDS and 1% HPCD in the presence of sterols, after which Fab fragments of either anti-MELADL or control antibody (IgG-R139) were microinjected into the cytoplasm (34). After 30 min (zero time), the coverslip was mounted into a square holder (Ludin chamber) and positioned on the microscope stage, after which the cells were switched to sterol-depleting imaging medium, and images were collected every 5 min for 75 min. Note that only images taken at 0, 30, and 60 min are presented. During the experiment, cells were kept at 37°C on a microscope enclosed in an environmental chamber. After imaging, the coverslip chamber was removed from the stage after which the cells were fixed in the chamber, permeabilized as described above, and stained with Alexa Fluor 594 conjugated goat anti-rabbit IgG (1:200 dilution). To identify injected cells, the coverslip chamber was mounted back on the microscope stage, which was repositioned to the stored x–y coordinates on the stage. This allowed us to image the same field of cells as detected during live cell imaging. (Scale bar, 25 µm.)

View larger version (21K): [in this window] [in a new window]   Fig. 3. Binding of Insig-1 to Scap does not block binding of antibody to the MELADL sequence. 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-SREBP-2 (all dishes) and 0.2 µg of pCMV–Scap and 0.05 µg of pCMV-Insig-1-Myc as indicated. Twelve hours after transfection, cells were switched to sterol-depleting medium containing 1% HPCD and incubated at 37°C for 1 h. The cells were then incubated with sterol-depleting medium (without HPCD) in the absence or presence of 1 µg/ml of 25-HC. After incubation for 3 h at 37°C, the cells were harvested, and aliquots of microsomal membranes (150 µg), in a final volume of 0.3 ml of Buffer B, were mixed with 3 µg of anti-MELADL antibody in the absence or presence of 0.5 mg of the MELADL-containing synthetic peptide as indicated. After incubation for 1 h at 4°C, the microsomal membranes were pelleted by centrifugation, solubilized in Buffer D (50 mM Hepes-KOH, pH 7.2/150 mM KCl/1 mM MgCl2, and a mixture of protease inhibitors; see Materials and Methods) supplemented with 0.1% Nonidet P-40, and incubated with Protein A/G beads for 30 min at 4°C. The resulting supernatant (S) and pellet (P) (10% of S) fractions were subjected to 8% SDS/PAGE and immunoblot analysis with IgG-9D5 (anti-Scap) and IgG-9E10 (anti-Insig-1).

View larger version (22K): [in this window] [in a new window]   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%.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Amino acid insertions or deletions adjacent to MELADL sequence alter Scap function. (A) Amino acid sequence of loop 6 of Scap, showing sites of insertions (arrows) and deletions (red box). (B and C) On day 0, Scap-deficient SRD-13A cells were set up in 60-mm dishes. (B) SREBP-2 cleavage. On day 2, cells were transfected with 2.5 µg of pTK-SREBP-2 and 0.2 µg of the indicated wild-type or mutant version of pCMV–Scap. Twelve hours after transfection, cells were switched to sterol-depleting medium with 1% HPCD and incubated for 1 h at 37°C. Cells were then incubated with sterol-depleting medium (without HPCD) for 3 h at 37°C and harvested for preparation of nuclear extract and membrane fractions. These fractions were subjected to 8% SDS/PAGE and immunoblot analysis with anti-HSV IgG (anti-SREBP-2) and IgG-R139 (anti-Scap) as indicated. (C) Flag-Sec23 pulldown. On day 2, cells were transfected with 0.4 µg of wild-type or the indicated mutant version of pCMV–Scap (i, insertion; d, deletion). Twelve hours after transfection, cells were treated the same as in B and harvested. Microsomal membranes (150 µg) were analyzed for Scap binding to COPII proteins using the Flag-Sec23 pull-down assay as described in Fig. 2A. Supernatant (S) and pellet (P) (5% of S) fractions were subjected to 8% SDS/PAGE and immunoblot analysis with IgG-R139 (anti-Scap).

View larger version (32K): [in this window] [in a new window]   Fig. 6. Differential mechanisms by which cholesterol and oxysterols trigger Insig binding to Scap, which in turn abrogates COPII binding to the MELADL sorting signal in Scap, inhibiting the ER-to-Golgi transport of SREBPs.

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