Cholesterol-induced conformational change in SCAP enhanced by Insig proteins and mimicked by cationic amphiphiles

Abstract

Sterols mediate feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway by preventing movement of the SREBP cleavage-activating protein (SCAP)/SREBP complex from endoplasmic reticulum (ER) to Golgi, where proteolytic cleavage of SREBPs releases the transcription factor domain that activates genes for lipid biosynthesis. Our laboratory previously used a trypsin cleavage assay to show that the conformation of SCAP is altered in vitro by addition of cholesterol to ER membranes. More recently, Insig-1 and Insig-2 were identified as ER resident proteins that bind the SCAP/SREBP complex and promote its ER retention when cells are treated with sterols. Here, we use the trypsin assay to show that Insig proteins reduce the concentration of cholesterol needed in vitro to produce the conformational change in SCAP. Insig-1 and Insig-2 also enhance the conformational change in SCAP that occurs upon addition of certain cationic amphiphiles, such as chlorpromazine, trifluoperazine, and imipramine, which mimic the effect of cholesterol. The effects of cationic amphiphiles raise the possibility that SCAP may monitor specifically the composition of the cytoplasmic leaflet of the ER membrane.

Fig. 1.

Insig-1 enhances SCAP's response to in vitro treatment with cholesterol. (A) SRD-13A cells were transfected with 2 μg of pCMV (cytomegalovirus)-SCAP and 2 μg of pTK-HSV-SREBP-2 in the absence or presence of 0.3 μg of pCMV-Insig-1-Myc as indicated. After the cells were harvested, aliquots of the 20,000 × g membrane suspension (100 μg) were incubated for 20 min at room temperature with the indicated concentration of cholesterol/MCD complex. At the end of the incubation, the membranes were treated sequentially with trypsin (30°C for the 30 min) and PNGase F and were then subjected to SDS/PAGE and immunoblot analysis with anti-SCAP IgG-9D5. (B) Quantification of Insig-1's effect on SCAP's response to cholesterol. The relative intensity of the upper and lower bands in A was quantified by densitometry. (C) Insig-1 does not enhance SCAP's sensitivity to epicholesterol or oxysterols. SRD-13A cells were transfected with 2 μg of pCMV-SCAP and 2 μg of pTK-HSV-SREBP-2 in the absence or presence of 0.3 μg of pCMV-Insig-1-Myc as indicated. Aliquots of the 20,000 × g membrane suspension were incubated with 80 μM of the indicated sterol/MCD complex. After sterol treatment, SCAP's conformation was analyzed as described in A.(D) Relative intensity of the upper and lower bands in C was quantitated by densitometry. Chol. cholesterol; epichol., epicholesterol; 25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol.

Fig. 2.

Both Insig-1 and Insig-2 enhance SCAP's response to cholesterol. SRD-13A cells were transfected with 2 μg of pCMV-SCAP, 2 μg of pTK-HSV-SREBP-2, and the indicated amounts of pCMV-Insig-1-Myc (A) or pCMV-Insig-2-Myc (B). Aliquots of 20,000 × g membrane suspension were incubated for 20 min at room temperature with the indicated concentration of cholesterol/MCD complex. After sterol treatment, SCAP's conformation was analyzed as described in Fig. 1. (C) Relative intensity of the upper and lower bands in A and B was quantified by densitometry.

Fig. 3.

Sterol-resistant mutant forms of SCAP show diminished Insig-1-dependent response to cholesterol. (A) SRD-13A cells were transfected with 2μg of the indicated SCAP construct plus 2 μg of pTK-HSV-SREBP-2 in the absence or presence of 0.1 μg of pCMV-Insig-1-Myc as indicated. Aliquots of the 20,000 × g membrane suspension were incubated for 20 min at room temperature with the indicated concentration of the cholesterol/MCD complex. After sterol treatment, SCAP's conformation was analyzed as described in Fig. 1. (B) Relative intensity of the top and bottom bands in A was quantified by densitometry. Data are from three independent experiments comparing wild-type SCAP to a SCAP mutant: wild type vs. L315F (circles), wild type vs. Y298C (triangles), and wild type vs. D443N (squares). The wild-type SCAP immunoblots in A are from the experiment comparing wild type to SCAP (L315F).

Fig. 4.

Insig-1 sensitizes wild-type but not sterol-resistant SCAP to chlorpromazine and imipramine. SRD-13A cells were transfected with 2 μg of wild type or mutant pCMV-SCAP and 2 μg of pTK-HSV-SREBP-2 in the absence or presence of 0.3 μg of pCMV-Insig-1-Myc as indicated. Aliquots of the 20,000 × g membrane suspension were incubated for 20 min at room temperature with the indicated concentration of chlorpromazine or imipramine. After treatment with drug, SCAP's conformation was analyzed as described in Fig. 1. (A) Effect of varying concentrations of chlorpromazine on wild-type SCAP. (B) Quantification of data in A by densitometry. (C) Effect of varying concentrations of imipramine on wild-type SCAP. (D) Quantification of data in C by densitometry. (E) Effect of varying concentrations of imipramine on sterol-resistant mutant forms of SCAP. (F) Relative intensity of the top and bottom bands in E was quantified by densitometry. Data are from three independent experiments comparing wild-type SCAP to a SCAP mutant: wild type vs. L315F (circles), wild type vs. Y298C (triangles), and wild type vs. D443N (squares). The wild-type SCAP immunoblots in E are from the experiment comparing wild type to SCAP (L315F).

Fig. 5.

Structure-function analysis of compounds that alter SCAP conformation. SRD-13A cells were transfected with 2 μg of pCMV-SCAP, 2 μg of pTK-HSV-SREBP-2, and 0.3 μg of pCMV-Insig-1. Aliquots of the 20,000 × g membrane suspension were incubated for 20 min at room temperature with 80 μM of cholesterol/MCD complex (C) or 100 μM of the indicated compound (numbered 1–19). Stock solutions of drugs were freshly prepared in water (1, 2, 4–6, 10–12, 14, 15, and 17–19) or ethanol (3, 7–9, 13, and 16). After treatment with drug, SCAP's conformation was analyzed by trypsin digestion and immunoblot analysis as described in Fig. 1. The first and last lanes in each gel represent membranes treated with water or ethanol, respectively, in the absence of drug. The percent lower band generated by each compound is shown above the compound's chemical structure and represents the average of at least three experiments.

Fig. 6.

Additive effects of Insig-1 and trifluoperazine (TFP) on SCAP's response to cholesterol. (A) SRD-13A cells were transfected with 2 μg of pCMV-SCAP and 2 μg of pTK-HSV-SREBP-2, in the absence or presence of 0.3 μg of pCMV-Insig-1-Myc as indicated. Aliquots of the 20,000 × g membrane suspension were incubated for 20 min at room temperature with the indicated concentration of cholesterol/MCD complex alone (○) or in the presence of TFP at 10 μM(•) or 20 μM(▴). After treatment, SCAP's conformation was analyzed as described in Fig. 1. (B) Relative intensity of the top and bottom bands in A was quantified by densitometry. The actual data in A and B were normalized in Lower as follows: for each cholesterol curve, the value for % lower band generated in the absence of cholesterol and in the presence of 0, 10, or 20 μM TFP was subtracted from all other points on the curve, after which the subtracted values for each curve were normalized to the maximal value at 80μM cholesterol, which was set at 100%.