Sequence alignment of Insig proteins and membrane topology of human Insig-1. (A) Sequence alignment of the membrane domain of Insig proteins was performed by clustalw program (DNASTAR, Madison, WI). Residues identical to human Insig-1 are shown in yellow, and two conserved motifs are indicated by the bars. GenBank accession nos. for human Insig-1, human Insig-2, hamster Insig-1, Xenopus Insig-1, zebrafish Insig-1, and Schizosaccharomyces pombe Insig are AY112745, AF527632, AF527628, BC076862, AF527626, and CAB41653, respectively. (B) Proposed membrane topology of Insig-1 (13). Asp-205 is shown in black. Residues other than Asp-205 in the two conserved motifs are shown in orange. The residue in human Insig-2 corresponding to Asp-205 in human Insig-1 is Asp-149 (9).

Mutant Insig-1(D205A) does not bind to Scap but does bind to VAP proteins. (A) On day 0, SRD-13A cells were set up at 3.5 × 10⁵ cells per 60-mm dish. On day 2, cells in each dish were transfected with 0.5 μg of pCMV-Scap and 0.1 μg of WT or 0.25 μg of D205A mutant pCMV-Insig1-Myc, as indicated. At 6 h after the transfection, cells were changed to sterol-depleting medium C. On day 3, cells were switched to fresh medium C in the absence (−) or presence (+) of 0.3 μg/ml 25-HC. After incubation for 5 h, cells were harvested, lysed, and immunoprecipitated (IP) with polyclonal anti-Myc to precipitate Insig-1. Pellets (representing 0.25 dish of cells) and supernatants (representing 0.05 dish of cells) of the immunoprecipitation were subjected to SDS/PAGE and immunoblot (IB) analysis. Filters were exposed to film for 2–60 s. (B) On day 0, CHO-7 cells were set up at 7 × 10⁵ cells per 60-mm dish. On day 1, cells in each dish were transfected with 1 μg of pCMV-VAP-A-HA, 0.3 μg of pCMV-VAP-B-HA, 0.1 μg of WT, or 0.2 μg of D205A mutant pCMV-Insig1-Myc, as indicated. On day 3, cells were harvested, lysed, and immunoprecipitated (IP) with polyclonal anti-HA to precipitate VAP proteins. Pellets (representing 0.25 dish of cells) and supernatants (representing 0.05 dish of cells) were subjected to SDS/PAGE and immunoblot analysis. Filters were exposed to film for 1–60 s.

Mutant Insig-1(D205A) is defective in accelerating the degradation of HMG CoA reductase in the presence of 25-HC or lanosterol. On day 0, CHO-K1 cells were set up at 5 × 10⁵ cells per 60-mm dish. On day 1, cells were transfected in A with 1 μg of pCMV-HMG-Red-T7(TM1–8) and the indicated amount of WT or D205A mutant pCMV-Insig1-Myc and in B with 1 μg of pCMV-HMG-Red-T7(TM1–8) and either 0.1 μg of WT or 0.3 μg of D205A mutant pCMV-Insig1-Myc. At 6 h after transfection, cells were changed to sterol-depleting medium C. On day 3, cells were switched to fresh medium C in the absence (−) or presence (+) of either 1 μg/ml 25-HC plus 10 mM mevalonate (A) or the indicated concentration of lanosterol plus 10 mM mevalonate (B). After incubation for 5 h, cells were harvested, and membrane fractions were subjected to SDS/PAGE and immunoblot analysis with anti-T7 mAb and IgG-9E10 to detect HMG CoA reductase and Insig-1, respectively. Filters were exposed to film for 5–30 s.

Mutant Insig-1(D205A) is defective in suppressing SREBP-2 cleavage. On day 0, SRD-13A cells were set up at 3.5 × 10⁵ cells per 60-mm dish. On day 2, cells in each dish were transfected with 0.4 μg of pCMV-Scap, 2 μg of pTK-HSV-SREBP-2, and 0.1 μg of WT or 0.3 μg of mutant pCMV-Insig1-Myc, as indicated. On day 3, cells were switched to medium C supplemented with 1% hydroxypropyl-β-cyclodextrin and incubated for 1 h at 37°C. Cells were then washed twice with PBS and switched to medium C supplemented either with (+) or without (−) 1 μg/ml 25-HC (A and C) or the indicated concentration of cholesterol complexed to methyl-β-cyclodextrin (B). After incubation for 5 h, cells were harvested, fractionated into membrane and nuclear extracts, and subjected to SDS/PAGE and immunoblot analysis. Membrane fractions were immunoblotted with anti-HSV, IgG-9E10, and IgG-R139 to detect precursor form of SREBP-2 (P), Insig-1, and Scap, respectively. The nuclear extracts were immunoblotted with anti-HSV to detect the nuclear form of SREBP-2. Filters were exposed to film for 5–30 s. Chol/MCD, cholesterol complexed to methyl-β-cyclodextrin.

Mutant Insig-1(D205A) is not stabilized by Scap. On day 0, SRD-13A cells were set up at 3.5 × 10⁵ cells per 60-mm dish. On day 2, cells were transfected with 1 μg of pTK-Scap and 0.2 μg of WT or D205A mutant pTK-Insig1-Myc, as indicated. At 6 h after transfection, cells were changed to sterol-depleting medium C. On day 3, cells were switched to fresh medium C in the absence (−) or presence (+) of 0.1 μg/ml 25-HC, with or without the addition of 10 μM MG-132. After incubation for 5 h, cells were harvested, and cell lysates were subjected to SDS/PAGE and immunoblot analysis with IgG-9E10 and IgG-R139 to detect Insig-1 and Scap, respectively. Filters were exposed to film for 10–60 s.

Mutant Insig-2(D149A) is defective in suppressing SREBP-2 cleavage and accelerating the degradation of HMG CoA reductase. (A) SRD-13A cells were set up, treated, and analyzed in the same manner as described in Fig. 2A except that 1 μg of WT or 3 μg of mutant pCMV-Insig2-Myc was transfected. (B) CHO-K1 cells were set up, treated, and analyzed in the same manner as described in Fig. 5. Filters were exposed to film for 5–30 s.