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Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

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Association of erlins with the SREBP regulatory machinery. (A) CoIP of SREBP pathway proteins with endogenous erlin-2 in lysates of HeLa cells grown with complete (F) or LD medium. Samples were probed by Western blotting as indicated. CANX, calnexin. 10 times more cell equivalents were loaded in IP lanes compared with input, except for erlin-2 blot (1 equivalent). Western blot images are typical of three separate repeats. Numbers below the panel indicate LD/F ratios of IP target proteins averaged from three experiments. P-values for LD versus complete (F): Scap, Srebp-1, and Srebp-2, <3.5 × 10−4; erlin-2 and gp78, >0.61. (B) Diagram of the inducible erlin clustering system. (C) Fluorescence images showing Insig localization to induced erlin clusters. Cells expressing the indicated combinations of recombinant proteins were incubated without (left column) or with (all other columns) rapamycin. Corresponding GFP and red mCherry fluorescent images of a representative cell from each condition are shown in grayscale. Arrows indicate some Insig-1-mCherry foci colocalized with erlin-GFP foci. (D) CoIP of recombinant erlin and SREBP–Scap–Insig. Lysates of 293T cells expressing various combinations of erlin-2-V5, Insig-1-Myc, hamster Scap (cgScap), and FLAG-SREBP-2 were IP with anti-V5 antibody. Input (Inp.; 1 equivalent) and IPed material (IP; 10 equivalents, except for erlin-2 blot [1 equivalent]) was analyzed by Western blotting. Certain transfections (asterisk) received four times more Insig-1-Myc cDNA to compensate for the instability of Insig in the absence of overexpressed Scap (Gong et al., 2006). (E) CoIP of recombinant erlin and Scap with Insig-1 depletion. Experiments were performed as in D with the indicated sample depleted of endogenous Insig-1 with siRNA. Insig-1 mRNA levels determined by q-RT-PCR are shown in the bar graph (bottom). Error bars indicate standard deviations.
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fig3: Association of erlins with the SREBP regulatory machinery. (A) CoIP of SREBP pathway proteins with endogenous erlin-2 in lysates of HeLa cells grown with complete (F) or LD medium. Samples were probed by Western blotting as indicated. CANX, calnexin. 10 times more cell equivalents were loaded in IP lanes compared with input, except for erlin-2 blot (1 equivalent). Western blot images are typical of three separate repeats. Numbers below the panel indicate LD/F ratios of IP target proteins averaged from three experiments. P-values for LD versus complete (F): Scap, Srebp-1, and Srebp-2, <3.5 × 10−4; erlin-2 and gp78, >0.61. (B) Diagram of the inducible erlin clustering system. (C) Fluorescence images showing Insig localization to induced erlin clusters. Cells expressing the indicated combinations of recombinant proteins were incubated without (left column) or with (all other columns) rapamycin. Corresponding GFP and red mCherry fluorescent images of a representative cell from each condition are shown in grayscale. Arrows indicate some Insig-1-mCherry foci colocalized with erlin-GFP foci. (D) CoIP of recombinant erlin and SREBP–Scap–Insig. Lysates of 293T cells expressing various combinations of erlin-2-V5, Insig-1-Myc, hamster Scap (cgScap), and FLAG-SREBP-2 were IP with anti-V5 antibody. Input (Inp.; 1 equivalent) and IPed material (IP; 10 equivalents, except for erlin-2 blot [1 equivalent]) was analyzed by Western blotting. Certain transfections (asterisk) received four times more Insig-1-Myc cDNA to compensate for the instability of Insig in the absence of overexpressed Scap (Gong et al., 2006). (E) CoIP of recombinant erlin and Scap with Insig-1 depletion. Experiments were performed as in D with the indicated sample depleted of endogenous Insig-1 with siRNA. Insig-1 mRNA levels determined by q-RT-PCR are shown in the bar graph (bottom). Error bars indicate standard deviations.

Mentions: To determine whether the SREBP regulatory machinery is physically associated with erlins, we analyzed immunoprecipitates (IPs) of endogenous erlins from cell lysates (Fig. 3 A). Under conditions of cholesterol sufficiency, we found that Insig-1, Scap, SREBP-1a, and SREBP-2 all coimmunoprecipitated (coIPed) with erlin-2 (Fig. 3 A) and the associated erlin-1 (Fig. S3 B). We also detected coIP of the ubiquitin E3 ligase gp78 with erlin-2 as reported previously (Pearce et al., 2007; Jo et al., 2011). The coIP of these proteins with erlins was specific because three other transmembrane proteins of the ER (derlin-1, Sec61α, and calnexin) as well as the peripheral ER protein p97 were not detectable in erlin IPs (Fig. 3 A). Moreover, after cholesterol depletion, Insig-1, Scap, SREBP-1a, and SREBP-2 were undetectable or strongly diminished in erlin IPs (Fig. 3 A). However, gp78 association was unchanged as seen before (Pearce et al., 2007; Jo et al., 2011). This supports the model that a physical association of erlins with SREBP–Scap–Insig is functionally relevant to regulation of the SREBP pathway.


Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

Association of erlins with the SREBP regulatory machinery. (A) CoIP of SREBP pathway proteins with endogenous erlin-2 in lysates of HeLa cells grown with complete (F) or LD medium. Samples were probed by Western blotting as indicated. CANX, calnexin. 10 times more cell equivalents were loaded in IP lanes compared with input, except for erlin-2 blot (1 equivalent). Western blot images are typical of three separate repeats. Numbers below the panel indicate LD/F ratios of IP target proteins averaged from three experiments. P-values for LD versus complete (F): Scap, Srebp-1, and Srebp-2, <3.5 × 10−4; erlin-2 and gp78, >0.61. (B) Diagram of the inducible erlin clustering system. (C) Fluorescence images showing Insig localization to induced erlin clusters. Cells expressing the indicated combinations of recombinant proteins were incubated without (left column) or with (all other columns) rapamycin. Corresponding GFP and red mCherry fluorescent images of a representative cell from each condition are shown in grayscale. Arrows indicate some Insig-1-mCherry foci colocalized with erlin-GFP foci. (D) CoIP of recombinant erlin and SREBP–Scap–Insig. Lysates of 293T cells expressing various combinations of erlin-2-V5, Insig-1-Myc, hamster Scap (cgScap), and FLAG-SREBP-2 were IP with anti-V5 antibody. Input (Inp.; 1 equivalent) and IPed material (IP; 10 equivalents, except for erlin-2 blot [1 equivalent]) was analyzed by Western blotting. Certain transfections (asterisk) received four times more Insig-1-Myc cDNA to compensate for the instability of Insig in the absence of overexpressed Scap (Gong et al., 2006). (E) CoIP of recombinant erlin and Scap with Insig-1 depletion. Experiments were performed as in D with the indicated sample depleted of endogenous Insig-1 with siRNA. Insig-1 mRNA levels determined by q-RT-PCR are shown in the bar graph (bottom). Error bars indicate standard deviations.
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Related In: Results  -  Collection

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fig3: Association of erlins with the SREBP regulatory machinery. (A) CoIP of SREBP pathway proteins with endogenous erlin-2 in lysates of HeLa cells grown with complete (F) or LD medium. Samples were probed by Western blotting as indicated. CANX, calnexin. 10 times more cell equivalents were loaded in IP lanes compared with input, except for erlin-2 blot (1 equivalent). Western blot images are typical of three separate repeats. Numbers below the panel indicate LD/F ratios of IP target proteins averaged from three experiments. P-values for LD versus complete (F): Scap, Srebp-1, and Srebp-2, <3.5 × 10−4; erlin-2 and gp78, >0.61. (B) Diagram of the inducible erlin clustering system. (C) Fluorescence images showing Insig localization to induced erlin clusters. Cells expressing the indicated combinations of recombinant proteins were incubated without (left column) or with (all other columns) rapamycin. Corresponding GFP and red mCherry fluorescent images of a representative cell from each condition are shown in grayscale. Arrows indicate some Insig-1-mCherry foci colocalized with erlin-GFP foci. (D) CoIP of recombinant erlin and SREBP–Scap–Insig. Lysates of 293T cells expressing various combinations of erlin-2-V5, Insig-1-Myc, hamster Scap (cgScap), and FLAG-SREBP-2 were IP with anti-V5 antibody. Input (Inp.; 1 equivalent) and IPed material (IP; 10 equivalents, except for erlin-2 blot [1 equivalent]) was analyzed by Western blotting. Certain transfections (asterisk) received four times more Insig-1-Myc cDNA to compensate for the instability of Insig in the absence of overexpressed Scap (Gong et al., 2006). (E) CoIP of recombinant erlin and Scap with Insig-1 depletion. Experiments were performed as in D with the indicated sample depleted of endogenous Insig-1 with siRNA. Insig-1 mRNA levels determined by q-RT-PCR are shown in the bar graph (bottom). Error bars indicate standard deviations.
Mentions: To determine whether the SREBP regulatory machinery is physically associated with erlins, we analyzed immunoprecipitates (IPs) of endogenous erlins from cell lysates (Fig. 3 A). Under conditions of cholesterol sufficiency, we found that Insig-1, Scap, SREBP-1a, and SREBP-2 all coimmunoprecipitated (coIPed) with erlin-2 (Fig. 3 A) and the associated erlin-1 (Fig. S3 B). We also detected coIP of the ubiquitin E3 ligase gp78 with erlin-2 as reported previously (Pearce et al., 2007; Jo et al., 2011). The coIP of these proteins with erlins was specific because three other transmembrane proteins of the ER (derlin-1, Sec61α, and calnexin) as well as the peripheral ER protein p97 were not detectable in erlin IPs (Fig. 3 A). Moreover, after cholesterol depletion, Insig-1, Scap, SREBP-1a, and SREBP-2 were undetectable or strongly diminished in erlin IPs (Fig. 3 A). However, gp78 association was unchanged as seen before (Pearce et al., 2007; Jo et al., 2011). This supports the model that a physical association of erlins with SREBP–Scap–Insig is functionally relevant to regulation of the SREBP pathway.

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Show MeSH
Related in: MedlinePlus