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. 1998 Oct 27;95(22):12848-53.
doi: 10.1073/pnas.95.22.12848.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

A Nohturfft  1 , M S Brown, J L Goldstein
Affiliations

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

A Nohturfft et al. Proc Natl Acad Sci U S A. .

Abstract

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

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Figures

Figure 1
Figure 1
Immunoblot analysis of SREBP-1 and SREBP-2 in wild-type CHO-7 and mutant 25-hydroxycholesterol-resistant cells. On day 0, the indicated cell line was set up in medium A supplemented with 5% lipoprotein-deficient serum. On day 2, the cells were switched to medium A containing 5% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.1% (vol/vol) ethanol containing the indicated final concentration of 25-hydroxycholesterol (25-OH Chol.). On day 3, the cells were harvested and fractionated into nuclear extract and 105 g membrane fraction as described (21, 29). Aliquots of the fractions (40 μg protein) were subjected to 7% SDS/PAGE and transferred to nitrocellulose. Immunoblot analysis was carried out with 10 μg/ml of IgG-2A4 (A) or 2 μg/ml of IgG-7D4 (B). Filters were exposed to film for 3 min (A, Upper), 45 s (A, Lower), 2 s (B, Upper), or 10 s (B, Lower). N and P denote the nuclear and precursor forms of SREBPs, respectively. X denotes a cross-reacting protein of unknown identity.
Figure 2
Figure 2
Comparison of sterol-sensing domains in SCAP, HMG CoA reductase, NPC1, Patched, and TRC8. Sequences are from Chinese hamster SCAP (amino acids 280–446), Chinese hamster HMG CoA reductase (amino acids 57–224), mouse NPC1 (amino acids 617–791), mouse Patched (amino acids 420–589), and human TRC8 (amino acids 16–182). GenBank accession numbers are U67060, L00165, AF003348, U46155, and AF064801, respectively. Sequences were aligned with pileup from the Genetics Computer Group Sequence Analysis Software Package, version 8.0. Residues identical in at least three of the five proteins are highlighted in black. Residues chemically similar in at least four of the five proteins are boxed. Overbars denote positions of transmembrane domains 2–6 in HMG CoA reductase (10). ∗ denote positions of point mutations in hamster SCAP (Y298C and D443N).
Figure 3
Figure 3
Activity of mutant SCAP(Y298C) in transfected cells. (A and B) Stimulation of cleavage of SREBPs by mutant SCAP(Y298C) in transfected 293 cells grown in the presence of sterols. On day 2 of growth, 293 cells were cotransfected with 9 μg of either pTK-HSV-BP1a encoding HSV epitope-tagged human SREBP-1a (A, lanes 3–13) or pTK-HSV-BP2 (B, lanes 3–13) together with the indicated amount of epitope-tagged wild-type SCAP (lanes 5, 8, and 11), SCAP(D443N) (lanes 6, 9, and 12), or SCAP(Y298C) (lanes 7, 10, and 13). The total amount of DNA in each dish was adjusted to 10 μg by addition of pTK empty vector. Three hours after transfection, cells were switched to medium B containing 10% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence (− sterols) or presence (+ sterols) of 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml of cholesterol as indicated. On day 3, 25 μg/ml of N-acetyl-leucinol-leucinol-norleucinol (ALLN) was added directly to the medium 4 h before cell harvesting and fractionation, which was carried out as described in Fig. 1. Aliquots of nuclear extract (A, 80 μg and B, 55 μg) and membranes (A, 100 μg and B, 110 μg) were subjected to 7% SDS/PAGE, transferred to nitrocellulose, and immunoblotted with either 0.5 μg/ml of IgG-HSV-Tag antibody (nuclear extracts) or 10 μg/ml of IgG-9D5 (membranes). Filters were exposed to film for 5 min (A), 30 s (B, Upper), and 50 s (B, Lower). N denotes the nuclear cleaved form of SREBPs. (C) Growth of CHO-7 cells. On day 0, CHO-7 cells were set up (5 × 105 cells/100-mm dish) in medium C (medium A containing 5% lipoprotein-deficient serum). On day 2, cells were transfected with either no plasmid, empty vector pTK, pTK3-SCAP encoding wild-type SCAP, pTK3-SCAP(D443N), or pTK3-SCAP(Y298C). All plasmids contained the G418-resistance gene neo. Three hours after transfection, cells were washed twice with PBS and refed with medium C. On day 3, cells were switched to medium D (medium C containing 0.75 mg/ml of G418) and refed every 2–3 days. No cells survived in dishes mock-transfected without any plasmid. On day 14, all surviving cells from duplicate, vector-transfected dishes were trypsinized, pooled, and set up in duplicate dishes in medium D (1.5 × 105 cells/60-mm dish). On day 15, cells were refed with medium D (Upper) or medium D containing 0.3 μg/ml of 25-hydroxycholesterol (Lower), and refed every 2–3 days. On day 18 (Upper) and day 22 (Lower), cells were fixed in 1% (vol/vol) glutaraldehyde, stained with crystal violet, and photographed.
Figure 4
Figure 4
Sterols alter endo H sensitivity of SCAP. The diagram shows a schematic of the domain structure of SCAP, denoting the approximate position of the protease-resistant fragment recognized by mAb IgG-9D5. Numbers below the diagram denote sites of N-linked glycosylation (5). On day 0, CHO-7 cells were set up in medium A supplemented with 10% fetal calf serum and 50 μg protein/ml of low density lipoprotein. On day 2, cells were switched to medium A containing 10% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.1% ethanol containing the indicated final concentration of 25-hydroxycholesterol (25-OH Chol.). After incubation for 16 h, cells were harvested, and membrane fractions were prepared as described in Materials and Methods. Aliquots of the membrane fraction (42 μg protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–16) of 17 μg/ml of trypsin. Proteolysis was stopped, and the samples were incubated for 16 h at 37°C in the absence (lanes 1–8) or presence (lanes 9–16) of the indicated glycosidase, subjected to SDS/PAGE, transferred to nitrocellulose, and immunoblotted with 10 μg/ml of IgG-9D5. The filter was exposed to film for 1.5 min. Numbers 1–3 on the right denote differentially glycosylated forms of the protease-resistant SCAP fragment containing either two (band 1), one (band 2), or no (band 3) N-linked oligosaccharides (5).
Figure 5
Figure 5
Regulated endo H sensitivity of SCAP is perturbed in 25-hydroxycholesterol-resistant CHO cell lines that harbor mutations in SCAP or SREBP-2. On day 0, the indicated cells were set up in medium A supplemented with 10% fetal calf serum. On day 2, cells were switched to medium A containing 10% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in either the absence (− sterols) or presence (+ sterols) of 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml of cholesterol as indicated. After incubation for 16 h, cells were harvested, and membrane fractions were prepared as described in Materials and Methods. Aliquots of membranes (A, 50 μg and B, 54 μg) were incubated in the absence or presence of 17 μg/ml of trypsin as indicated. Proteolysis was stopped, and the samples were incubated at 37°C for 16 h either in the absence (lanes 1–12) or presence (lanes 13–18) of endo H, subjected to SDS/PAGE, and transferred to nitrocellulose. Filters were blotted with 10 μg/ml of IgG-9D5 and exposed to film for 2 min (A) and 20 sec (B). ∗ denotes a cross-reacting protein of unknown identity. Numbers 1–3 on the right denote differentially N-glycosylated forms of the protease-resistant SCAP fragment as described in the legend to Fig. 4.
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