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Proc Natl Acad Sci U S A. Apr 18, 2006; 103(16): 6154–6159.
Published online Apr 10, 2006. doi:  10.1073/pnas.0601923103
PMCID: PMC1435366
Biochemistry

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis

Abstract

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells.

Keywords: 25-hydroxycholesterol, lanosterol, membrane proteins, proteolytic activation, Scap

Insig-1 and Insig-2 are closely related polytopic membrane proteins of the endoplasmic reticulum (ER). They regulate cholesterol homeostasis by binding to two ER proteins, 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and Scap, in a sterol-dependent manner (1). HMG CoA reductase catalyzes the rate-limiting step in the synthesis of cholesterol (2). Sterols stimulate its binding to either Insig-1 or Insig-2, which is an event that leads to the ubiquitination of reductase by an Insig-bound ubiquitin ligase (gp78) (3). Ubiquitinated reductase is destroyed rapidly in proteasomes (4, 5).

Scap is an escort protein for sterol regulatory element-binding proteins (SREBPs) (6), which are membrane-bound transcription factors that activate transcription of all of the known genes encoding cholesterol biosynthetic enzymes (7). In sterol-depleted cells, Scap transports SREBPs from the ER to the Golgi apparatus, where SREBPs undergo proteolytic activation. The accumulation of cholesterol triggers binding of Scap to either Insig-1 or Insig-2, which is a reaction that leads to the ER retention of Scap and prevents the delivery of its bound SREBPs to the Golgi apparatus (8, 9). Besides blocking the proteolytic activation of SREBPs, the interaction between Scap and Insig-1 also leads to the stabilization of Insig-1, which otherwise is degraded rapidly by proteasomes (10).

Scap and HMG CoA reductase share sterol-sensing domains that mediate the binding of both proteins to Insig-1 and Insig-2 (1). Mutations in the sterol-sensing domains of Scap affect the proteolytic processing of SREBPs. One class of mutations (which includes D443N, Y298C, and L315F) disrupts Scap–Insig binding (8, 11). Cells that express any one of these mutant Scap proteins overproduce cholesterol because of the inability to retain Scap in the ER when cholesterol accumulates. D428A, another substitution mutation, has the opposite effect. This substitution enhances the affinity of the Scap–Insig interaction (12). As a result, Scap(D428A) fails to transport SREBPs to the Golgi apparatus, even under conditions of sterol depletion.

Unlike Scap, we know little regarding the crucial residues in Insig-1 and Insig-2 that are required for their function. In these studies, we identify Asp-205 in Insig-1 and the corresponding Asp-149 in Insig-2 as essential residues that are required for interacting with Scap and HMG CoA reductase. This aspartic acid is immediately adjacent to the fourth transmembrane helix of Insig-1 or Insig-2 at the cytosolic side of the ER membrane. When this aspartic acid is mutated to alanine, the mutant Insig protein fails to inhibit the sterol-induced, Scap-mediated proteolytic processing of SREBPs, and it also fails to accelerate the sterol-induced, proteasomal degradation of HMG CoA reductase in cells that are loaded with sterols.

Results

Fig. 1A shows a sequence alignment of the membrane-spanning domains of Insig proteins in species ranging from human to yeast. Two regions were found to be conserved in all Insig proteins that were analyzed. One of the most highly conserved regions is the WXXFDRSR sequence, which is located at the junction of the fourth transmembrane helix and the adjacent cytosolic loop. The other is the GNXGRXL motif, which is located at the junction of sixth transmembrane helix and the cytoplasmic tail (Fig. 1B). To determine whether amino acid residues in these regions are important for the function of human Insig-1, we mutated each of the conserved residues in these two motifs individually to alanine and assayed the ability of the mutant Insig-1 to block processing of SREBP-2 in cultured hamster cells. Although some of these substitutions had partial effects, only one caused a complete loss of function. This one was the substitution of alanine for Asp-205, which is the most NH2-terminal residue in the third cytosolic loop according to the proposed membrane topology of Insig-1 (13).

Fig. 1.
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, ...

Fig. 2A shows an experiment in which plasmids encoding epitope-tagged SREBP-2, Scap, and WT or D205A mutant human Insig-1 were transfected into SRD-13A cells (a line of mutant CHO cells that are deficient in Scap) (14). When SREBP-2 was cotransfected with Scap, the cleaved NH2-terminal fragment of SREBP-2 appeared in nuclear extracts. The appearance of nuclear SREBP-2 was not blocked by 25-hydroxycholesterol (25-HC) (lanes 1 and 2), which is a sterol that potently inhibits the cleavage of SREBP-2 in nontransfected cells. We have shown (8) that this failure is caused by the molar excess of transfected Scap over endogenous Insig, which leaves a portion of Scap free to transport SREBP-2 to the Golgi apparatus even in the presence of 25-HC. When the cells were transfected with a cDNA encoding WT Insig-1, the ability of 25-HC to block SREBP-2 cleavage was restored (lane 4). When the D205A mutant of Insig-1 was transfected, 25-HC remained unable to block SREBP-2 cleavage (lane 6). Similar results were obtained when cholesterol was delivered to the cells as a complex with methyl-β-cyclodextrin (Fig. 2B, lanes 4–6 and 8–10).

Fig. 2.
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, ...

To determine whether the defect in Insig-1(D205A) is due to the loss of the negative charge, we prepared plasmids encoding Insig-1 with various substitutions at this position and transfected them into Scap-deficient SRD-13A cells, together with cDNAs encoding SREBP-2 and Scap (Fig. 2C). Substitution of glutamic acid for aspartic acid preserved the ability of Insig-1 to block SREBP-2 cleavage in the presence of 25-HC (Fig. 2C, lanes 7 and 8). Substitution with alanine or arginine destroyed this function (Fig. 2C, lanes 5–6 and 9–10). These data suggest that Insig-1 requires a negatively charged amino acid at position 205 to perform its function.

Insig-1 inhibits SREBP processing by means of sterol-dependent binding to Scap (8). The experiments shown in Fig. 3A were designed to determine whether Insig-1(D205A) is impaired in its binding to Scap. For this purpose, we transfected plasmids encoding Scap and Myc-tagged WT Insig-1 or the D205A mutant into SRD-13A cells. After incubation in the presence or absence of 25-HC, cells were harvested, and the cell lysates were incubated with anti-Myc to precipitate Insig-1. Supernatants and precipitates were subjected to SDS/PAGE. Insig-1 and Scap were visualized by immunoblotting. As shown in Fig. 3A, Scap was coimmunoprecipitated with WT Insig-1 in the presence of 25-HC (lane 5). It was not coimmunoprecipitated with Insig-1(D205A) regardless of the presence of 25-HC (lanes 6 and 7). In the same experiment, we also examined the binding of Insig-1 to vesicle-associated membrane protein-associated protein (VAP)-A and VAP-B proteins, which are two abundant membrane proteins of the ER and Golgi apparatus that bind to various native membrane proteins (15, 16) and have been shown (8) to copurify with Scap. As shown in Fig. 3B, both WT Insig-1 and the D205A mutant of Insig-1 were coimmunoprecipitated with VAP-A and VAP-B. The binding between Insig-1 and VAP proteins was not regulated by sterols (data not shown). These data indicate that the D205A substitution disrupts selectively the binding of Insig-1 to Scap but not to VAP proteins.

Fig. 3.
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 ...

We have shown (10) that free Insig-1 is degraded rapidly and that it is stabilized when it binds to Scap. Because Insig-1(D205A) does not bind to Scap, it is predicted to undergo rapid degradation in the absence or presence of sterols. Fig. 4 shows an experiment in which we transfected SRD-13A cells with a plasmid encoding WT or the D205A mutant of Insig-1 that is driven by the weak thymidine kinase (TK) promoter. In the absence of Scap, neither WT nor mutant Insig-1 was detectable by immunoblotting in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 25-HC. Both proteins were visualized only when proteasomal degradation was blocked by MG-132 (lanes 4 and 7). When Scap was cotransfected and the cells were incubated with 25-HC, WT Insig-1 became detectable in the absence of MG-132 (lane 9). In contrast, Insig-1(D205A) was not stabilized by Scap plus 25-HC (lane 11).

Fig. 4.
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 ...

Fig. 5 shows experiments in which we examined whether the D205A mutation also abolishes the ability of Insig-1 to accelerate the degradation of HMG CoA reductase. Studies have shown (5, 17) that Insig-mediated degradation is triggered by the combined action of a sterol (25-HC or lanosterol) and a nonsterol isoprenoid (geranylgeraniol) derived from mevalonate. We cotransfected WT CHO-K1 cells with a plasmid encoding WT or the D205A mutant of Insig-1 together with a plasmid encoding the NH2-terminal membrane-spanning domain of HMG CoA reductase [HMG CoA reductase(TM1-8)]. When HMG CoA reductase(TM1-8) was transfected alone, the amount of protein did not change after 5 h when 25-HC plus mevalonate were present (Fig. 5A, lanes 1 and 2). Like overexpressed Scap, the overexpressed reductase saturates endogenous Insig proteins, and the free reductase is not subjected to sterol-accelerated degradation (5). Cotransfection of WT Insig-1 restored the sterol-accelerated degradation of reductase in a dose-dependent manner (lanes 4 and 6). Cotransfection of Insig-1(D205A) did not accelerate the degradation of reductase in the presence of 25-HC plus mevalonate, even when we transfected a large excess of the plasmid encoding the mutant Insig-1 as compared with the WT (lanes 9–12). Similar results were obtained when we triggered the degradation of reductase with lanosterol instead of 25-HC (Fig. 5B). These data indicate that the D205A abolishes the ability of Insig-1 to accelerate the degradation of HMG CoA reductase.

Fig. 5.
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 ...

In Insig-2, the residue corresponding to Asp-205 is Asp-149 (Fig. 1A). As shown in Fig. 6, when we changed Asp-149 in Insig-2 to alanine, the protein lost the ability to suppress the cleavage of SREBP-2 (Fig. 6A) and accelerate the degradation of HMG CoA reductase (Fig. 6B) in cells that were loaded with sterols.

Fig. 6.
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 ...

Discussion

These studies identify Asp-205 in Insig-1 and the corresponding Asp-149 in Insig-2 as crucial participants in the mechanisms by which Insig proteins regulate sterol metabolism. When this aspartic acid in Insig-1 was mutated to alanine, Insig-1 no longer bound to Scap (Fig. 3A), and the mutant protein was ineffective in inhibiting SREBP-2 cleavage in cells loaded with either 25-HC or cholesterol (Fig. 2). The same mutation impaired the function of Insig-1 in accelerating the 25-HC or lanosterol-stimulated degradation of HMG CoA reductase (Fig. 5). Substitution of the corresponding Asp-149 in Insig-2 with alanine also destroyed the ability of Insig-2 to inhibit the processing of SREBP-2 and accelerate the degradation of HMG CoA reductase (Fig. 6).

The precise role of Asp-205 in Insig-1 and Asp-149 in Insig-2 is unknown. However, the change in the behavior of Insig-1(D205A) is unlikely to be caused by global unfolding, as shown by the normal association of this mutant protein with VAP-A and VAP-B (Fig. 3B), which do not contain a sterol-sensing domain and presumably bind to Insig proteins by means of some other mechanism.

The crucial aspartic acid identified in this study belongs to a conserved tetrapeptide sequence, DRSR (Fig. 1). Remarkably, this same tetrapeptide is also important in SREBPs, where it defines the boundary between the first transmembrane helix and its cytosolic interface. When the DRSR sequence in SREBP-2 was changed to AS, the intramembrane cleavage of SREBP-2 mediated by Site-2 protease was abolished (18). The DRSR sequences in Insig proteins and SREBPs may dictate the conformation of the protein at the surface of the membrane bilayers. The aspartic acid in the tetrapeptide may bind to the positively charged head groups of phospholipids to stabilize the correct folding of the transmembrane helix (19). Although the residues surrounding Asp-205 (FDRSR) are conserved in all Insig proteins, the aspartic acid residue appears to be the only one that is essential for function. Single substitution of the other residues with alanine produced only partial defects in the ability of Insig-1 to suppress SREBP-2 cleavage. Detailed structural studies of Insig proteins by crystallography or NMR may be useful for a better understanding of the functional role of the crucial aspartic acid that was identified in these studies.

Materials and Methods

Materials.

We obtained the following materials: MG-132 and Nonidet P-40 (Calbiochem), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO; Anatrace, Maumee, OH), sterols (Steraloids, Newport, RI), and hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin (Cyclodextrin Technologies Development, High Springs, FL). Lipoprotein-deficient serum (d > 1.215 g/ml) was prepared by ultracentrifugation of newborn calf serum (20). Solutions of sodium compactin, sodium mevalonate, and sodium oleate were prepared as described in refs. 21 and 22. Complexes of cholesterol with methyl-β-cyclodextrin were prepared as described in ref. 23.

Abs.

We obtained the following Abs: horseradish peroxidase-conjugated, donkey anti-mouse, and anti-rabbit IgGs (affinity-purified) (Jackson ImmunoResearch); anti-herpes simplex virus (HSV)-Tag (IgG) and anti-T7 (IgG) mAbs (Novagen); anti-hemagglutinin (HA) IgG polyclonal Ab (Santa Cruz Biotechnology); anti-Myc IgG polyclonal Ab (Bethyl Laboratories, Montgomery, TX); anti-HA mAb (Sigma); and cells producing IgG-9E10, which is a mouse mAb against Myc tag (American Type Culture Collection). IgG-9D5, which is a mouse mAb against hamster Scap, and IgG-R139, which is a rabbit polyclonal Ab against hamster Scap, were described in ref. 24.

Plasmid Constructs.

The following recombinant plasmids have been described: pCMV-Insig-1-Myc, encoding human Insig-1, followed by six tandem copies of a c-Myc epitope tag under control of the CMV promoter (8); pTK-Insig-1-Myc, encoding human Insig-1, followed by six tandem copies of a c-Myc epitope tag under control of the TK promoter (10); pCMV-Insig-2-Myc, encoding human Insig-2, followed by six tandem copies of a c-Myc epitope tag under control of CMV promoter (9); pCMV-Scap, encoding WT hamster Scap under control of CMV promoter (24); pTK-Scap, encoding WT hamster Scap under control of TK promoter (25); pCMV-HMG-Red(TM1–8)-T7, encoding amino acids 1–346 of hamster HMG-CoA reductase, followed by three tandem copies of the T7 epitope tag (MASMTGGQQMG) under control of CMV promoter (5). EST clones containing full-length human VAP-A (clone no. 328880) and VAP-B (clone no. 75005) cDNA were obtained from Invitrogen. pCMV-VAP-A-HA and pCMV-VAP-B-HA encode full-length human VAP-A and VAP-B, respectively, followed by three tandem copies of an HA epitope tag under control of CMV promoter. They were generated by ligating BamHI and AgeI-cleaved vector pcDNA3.1-(Myc)5 (26) with a PCR fragment that encodes full-length VAP-A or VAP-B with BamHI and NheI-cleaved overhangs, and an annealed double-stranded DNA oligonucleotide that encodes three tandem copies of a HA epitope with NheI- and AgeI-cleaved overhangs. Point mutations in Insig proteins were generated by site-directed mutagenesis with the QuikChange kit (Stratagene). The coding regions of all plasmids were sequenced before use.

Cell Culture.

Cells were maintained in monolayer culture at 37°C in 8–9% CO2. CHO-K1 cells were maintained in medium A (1:1 mixture of Ham's F-12 medium and DMEM supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin sulfate) supplemented with 5% (vol/vol) FCS. CHO-7 cells are a clone of CHO-K1 cells selected for growth in lipoprotein-deficient serum (27); they were maintained in medium A supplemented with 5% lipoprotein-deficient serum. SRD-13A cells are mutant CHO-7 cells deficient in Scap (14); they were maintained in medium B (medium A supplemented with 5% FCS/5 μg/ml cholesterol/1 mM sodium mevalonate/20 μM sodium oleate).

Transient Transfection, Cell Fractionation, and Immunoblot Analysis.

Cells were transiently transfected with FuGENE 6 reagent (Roche Applied Science) according to the manufacturer's protocol. The total amount of DNA in each transfection was adjusted to 3 μg per dish by addition of pcDNA3.1 (Invitrogen). After transfection, cells were incubated with sterol-depleting medium C (medium A supplemented with 5% lipoprotein-deficient serum, 50 μM sodium compactin, and 50 μM sodium mevalonate) and treated as described in the figure legends. At the end of the incubation, duplicate dishes of cells for each variable were harvested and pooled. They were fractionated into membrane, and nuclear extracts (18) or prepared as whole cell lysates (10) as described in the indicated reference. All fractions were subjected to SDS/PAGE and immunoblot analysis. The proteins were transferred to Hybond-C extra nitrocellulose filters (Amersham Biosciences), which were incubated with one of the following Abs: 1 μg/ml anti-Myc IgG-9E10, 2 μg/ml anti-Scap R139, anti-HSV (1:15,000 dilution), anti-T7 (1:1,000 dilution), or anti-HA mAb (1:2,000 dilution). Bound Abs were visualized with peroxidase-conjugated, affinity-purified donkey anti-mouse or anti-rabbit IgG (1:5,000 dilution) by using the SuperSignal CL-HRP substrate system (Pierce) according to the manufacturer's instructions. Gels were calibrated with prestained molecular-weight markers (New England Biolabs). Filters were exposed to Kodak X-Omat Blue XB-1 film at room temperature for the indicated time.

Immunoprecipitation.

The pooled cell pellets from duplicate dishes of cells were lysed in 0.5 ml of buffer A (50 mM Hepes·KOH, pH 7.6/100 mM NaCl/1.5 mM MgCl2/10 μg/ml leupeptin/5 μg/ml pepstatin A/25 μg/ml N-acetyl-leucyl-leucyl-l-norleucinal/2 μg/ml aprotinin) supplemented with 0.1% (vol/vol) Nonidet P-40 to immunoprecipitate Myc epitope-tagged Insig-1 or 0.5% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) to immunoprecipitate HA-tagged VAP proteins. Cell lysates were immunoprecipitated with 40 μg/ml anti-Myc or anti-HA polyclonal Ab together with 50 μl of protein A/G agarose beads (Santa Cruz Biotechnology), as described in ref. 24.

Acknowledgments

We thank our colleague Russell DeBose-Boyd for helpful suggestions and critical review of the manuscript and Lisa Beatty, Angela Carroll, and Marissa Hodgin for their invaluable help with tissue culture. This work was supported by National Institutes of Health Grant HL20948 and a grant from the Perot Family Foundation. J.Y. was supported by American Heart Association National Scientific Development Grant 0630029N.

Abbreviations

ER
endoplasmic reticulum
HA
hemagglutinin
25-HC
25-hydroxycholesterol
HMG
3-hydroxy-3-methylglutaryl
HSV
herpes simplex virus
SREBP
sterol regulatory element-binding protein
TK
thymidine kinase
VAP
vesicle-associated membrane protein-associated protein.

Footnotes

Conflict of interest statement: No conflicts declared.

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