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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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Cholesterol-dependent changes in diffusional mobility of erlin-2. (A) Confocal images during FRAP of a HeLa cell expressing erlin-2-GFP. Shown are images before fluorescence bleaching (pre-bleach) or at the indicated times after bleaching (red box). (B) Plot showing FRAP of erlin-2-GFP under conditions of cholesterol sufficiency (FBS; black), cholesterol depletion (LD; red), or cholesterol depletion followed by incubation with 50 µM cholesterol–β-MCD complex for 3 h (LD→chol.; green). Shown are averaged data from n = 7 cells. (C) Table summarizing erlin-2-GFP FRAP data. t1/2 and percentage values were calculated from two-phase association functions best describing the FRAP curves shown in B. (D) Plot of FRAP of LBR-GFP under lipid-sufficient (FBS; black) and lipid-depleted (LD; red) conditions; n = 5 cells. (E) Plot of FRAP of erlin-2-GFP under lipid-replete conditions without (FBS; black) or with tunicamycin (FBS+Tunicamycin; orange) to induce ER stress; n = 6 cells. Error bars indicate standard deviations.
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fig5: Cholesterol-dependent changes in diffusional mobility of erlin-2. (A) Confocal images during FRAP of a HeLa cell expressing erlin-2-GFP. Shown are images before fluorescence bleaching (pre-bleach) or at the indicated times after bleaching (red box). (B) Plot showing FRAP of erlin-2-GFP under conditions of cholesterol sufficiency (FBS; black), cholesterol depletion (LD; red), or cholesterol depletion followed by incubation with 50 µM cholesterol–β-MCD complex for 3 h (LD→chol.; green). Shown are averaged data from n = 7 cells. (C) Table summarizing erlin-2-GFP FRAP data. t1/2 and percentage values were calculated from two-phase association functions best describing the FRAP curves shown in B. (D) Plot of FRAP of LBR-GFP under lipid-sufficient (FBS; black) and lipid-depleted (LD; red) conditions; n = 5 cells. (E) Plot of FRAP of erlin-2-GFP under lipid-replete conditions without (FBS; black) or with tunicamycin (FBS+Tunicamycin; orange) to induce ER stress; n = 6 cells. Error bars indicate standard deviations.

Mentions: We used FRAP to examine whether the physical state of the erlin complex is sensitive to ER cholesterol levels (Fig. 5, A and B). We compared the diffusional mobility of erlin-2-GFP in cells grown in cholesterol replete (FBS) versus lipid-depleted conditions. In both cases, the FRAP recovery curves fit a two-phase exponential equation (r2 > 0.99), describing erlin-2 pools with “fast” and “slow” recovery kinetics (Fig. 5 C). In FBS, approximately half of the erlin-2-GFP was in a slow pool. After cholesterol depletion nearly all of it was in a slow pool, albeit with substantially slower recovery kinetics than the FBS slow pool. After restoration of cholesterol, both fast and slow pools of erlin-2-GFP had FRAP recovery times similar to cholesterol-replete cultures. Cholesterol-dependent changes in diffusional mobility were not observed for the model ER transmembrane protein laminin B receptor (LBR)–GFP (Ellenberg et al., 1997), showing that the effect on erlin-2 is selective (Fig. 5 D). Moreover, the effect cannot be ascribed to an ER stress response because there was no significant change in the mobility of erlin-2 in tunicamycin-treated cells (Fig. 5 E). Although we have not yet determined the molecular basis for the changes in erlin mobility with cholesterol depletion, our data nonetheless show that the physical properties of erlins are altered by standard conditions that induce SREBP pathway activation.


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

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

Cholesterol-dependent changes in diffusional mobility of erlin-2. (A) Confocal images during FRAP of a HeLa cell expressing erlin-2-GFP. Shown are images before fluorescence bleaching (pre-bleach) or at the indicated times after bleaching (red box). (B) Plot showing FRAP of erlin-2-GFP under conditions of cholesterol sufficiency (FBS; black), cholesterol depletion (LD; red), or cholesterol depletion followed by incubation with 50 µM cholesterol–β-MCD complex for 3 h (LD→chol.; green). Shown are averaged data from n = 7 cells. (C) Table summarizing erlin-2-GFP FRAP data. t1/2 and percentage values were calculated from two-phase association functions best describing the FRAP curves shown in B. (D) Plot of FRAP of LBR-GFP under lipid-sufficient (FBS; black) and lipid-depleted (LD; red) conditions; n = 5 cells. (E) Plot of FRAP of erlin-2-GFP under lipid-replete conditions without (FBS; black) or with tunicamycin (FBS+Tunicamycin; orange) to induce ER stress; n = 6 cells. Error bars indicate standard deviations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Cholesterol-dependent changes in diffusional mobility of erlin-2. (A) Confocal images during FRAP of a HeLa cell expressing erlin-2-GFP. Shown are images before fluorescence bleaching (pre-bleach) or at the indicated times after bleaching (red box). (B) Plot showing FRAP of erlin-2-GFP under conditions of cholesterol sufficiency (FBS; black), cholesterol depletion (LD; red), or cholesterol depletion followed by incubation with 50 µM cholesterol–β-MCD complex for 3 h (LD→chol.; green). Shown are averaged data from n = 7 cells. (C) Table summarizing erlin-2-GFP FRAP data. t1/2 and percentage values were calculated from two-phase association functions best describing the FRAP curves shown in B. (D) Plot of FRAP of LBR-GFP under lipid-sufficient (FBS; black) and lipid-depleted (LD; red) conditions; n = 5 cells. (E) Plot of FRAP of erlin-2-GFP under lipid-replete conditions without (FBS; black) or with tunicamycin (FBS+Tunicamycin; orange) to induce ER stress; n = 6 cells. Error bars indicate standard deviations.
Mentions: We used FRAP to examine whether the physical state of the erlin complex is sensitive to ER cholesterol levels (Fig. 5, A and B). We compared the diffusional mobility of erlin-2-GFP in cells grown in cholesterol replete (FBS) versus lipid-depleted conditions. In both cases, the FRAP recovery curves fit a two-phase exponential equation (r2 > 0.99), describing erlin-2 pools with “fast” and “slow” recovery kinetics (Fig. 5 C). In FBS, approximately half of the erlin-2-GFP was in a slow pool. After cholesterol depletion nearly all of it was in a slow pool, albeit with substantially slower recovery kinetics than the FBS slow pool. After restoration of cholesterol, both fast and slow pools of erlin-2-GFP had FRAP recovery times similar to cholesterol-replete cultures. Cholesterol-dependent changes in diffusional mobility were not observed for the model ER transmembrane protein laminin B receptor (LBR)–GFP (Ellenberg et al., 1997), showing that the effect on erlin-2 is selective (Fig. 5 D). Moreover, the effect cannot be ascribed to an ER stress response because there was no significant change in the mobility of erlin-2 in tunicamycin-treated cells (Fig. 5 E). Although we have not yet determined the molecular basis for the changes in erlin mobility with cholesterol depletion, our data nonetheless show that the physical properties of erlins are altered by standard conditions that induce SREBP pathway activation.

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