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The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Fryer LG, Jones B, Duncan EJ, Hutchison CE, Ozkan T, Williams PA, Alder O, Nieuwdorp M, Townley AK, Mensenkamp AR, Stephens DJ, Dallinga-Thie GM, Shoulders CC - J. Biol. Chem. (2013)

Bottom Line: The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi.The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation.We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo.

View Article: PubMed Central - PubMed

Affiliation: From the Endocrinology Centre, William Harvey Research Institute, Queen Mary University of London and Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom.

ABSTRACT
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

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Constitutively active Sar1B increases Srebp2 levels, whereas Sar1b deficiency decreases Srepb2 processing in McArdle-RH7777 cells.A–C, Srebp2 protein in stably overexpressing Sar1B:H79G and empty vector control cells. D–F, Sar1a and Sar1a+ Sar1b knockdown do not alter the levels of the 123-kDa precursor (D and F) or of the 68-kDa processed (E and F) Srebp2 protein. G–I, Sar1b knockdown decreases (H and I) the level of 68-kDa processed Srebp2 protein. A–I, data (mean ± S.E.) are from a minimum of three independent experiments. B, *, p < 0.05 versus cells with empty vector control. H, **, p < 0.01 versus scrambled control siRNA. In Western blot analyses, white line separators indicate that noncontiguous lanes from the same gel are shown. D–I, Scr, scrambled siRNA.
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Figure 6: Constitutively active Sar1B increases Srebp2 levels, whereas Sar1b deficiency decreases Srepb2 processing in McArdle-RH7777 cells.A–C, Srebp2 protein in stably overexpressing Sar1B:H79G and empty vector control cells. D–F, Sar1a and Sar1a+ Sar1b knockdown do not alter the levels of the 123-kDa precursor (D and F) or of the 68-kDa processed (E and F) Srebp2 protein. G–I, Sar1b knockdown decreases (H and I) the level of 68-kDa processed Srebp2 protein. A–I, data (mean ± S.E.) are from a minimum of three independent experiments. B, *, p < 0.05 versus cells with empty vector control. H, **, p < 0.01 versus scrambled control siRNA. In Western blot analyses, white line separators indicate that noncontiguous lanes from the same gel are shown. D–I, Scr, scrambled siRNA.

Mentions: We found that Sar1B:H79G overexpression led to a small, but nonsignificant, rise in the level of precursor Srebp2 (Fig. 6A) and a significant increase in the level of the 68-kDa processed polypeptide (Fig. 6, B and C). Levels of the control protein ATF6 were not altered (supplemental Fig. S3). Thus, these results indicate that there was sufficient endogenous Sar1 in the Sar1B:H79G cell lines (Fig. 2C) to support the ER export of both Srebp2 and ATF6 precursors.


The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Fryer LG, Jones B, Duncan EJ, Hutchison CE, Ozkan T, Williams PA, Alder O, Nieuwdorp M, Townley AK, Mensenkamp AR, Stephens DJ, Dallinga-Thie GM, Shoulders CC - J. Biol. Chem. (2013)

Constitutively active Sar1B increases Srebp2 levels, whereas Sar1b deficiency decreases Srepb2 processing in McArdle-RH7777 cells.A–C, Srebp2 protein in stably overexpressing Sar1B:H79G and empty vector control cells. D–F, Sar1a and Sar1a+ Sar1b knockdown do not alter the levels of the 123-kDa precursor (D and F) or of the 68-kDa processed (E and F) Srebp2 protein. G–I, Sar1b knockdown decreases (H and I) the level of 68-kDa processed Srebp2 protein. A–I, data (mean ± S.E.) are from a minimum of three independent experiments. B, *, p < 0.05 versus cells with empty vector control. H, **, p < 0.01 versus scrambled control siRNA. In Western blot analyses, white line separators indicate that noncontiguous lanes from the same gel are shown. D–I, Scr, scrambled siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924288&req=5

Figure 6: Constitutively active Sar1B increases Srebp2 levels, whereas Sar1b deficiency decreases Srepb2 processing in McArdle-RH7777 cells.A–C, Srebp2 protein in stably overexpressing Sar1B:H79G and empty vector control cells. D–F, Sar1a and Sar1a+ Sar1b knockdown do not alter the levels of the 123-kDa precursor (D and F) or of the 68-kDa processed (E and F) Srebp2 protein. G–I, Sar1b knockdown decreases (H and I) the level of 68-kDa processed Srebp2 protein. A–I, data (mean ± S.E.) are from a minimum of three independent experiments. B, *, p < 0.05 versus cells with empty vector control. H, **, p < 0.01 versus scrambled control siRNA. In Western blot analyses, white line separators indicate that noncontiguous lanes from the same gel are shown. D–I, Scr, scrambled siRNA.
Mentions: We found that Sar1B:H79G overexpression led to a small, but nonsignificant, rise in the level of precursor Srebp2 (Fig. 6A) and a significant increase in the level of the 68-kDa processed polypeptide (Fig. 6, B and C). Levels of the control protein ATF6 were not altered (supplemental Fig. S3). Thus, these results indicate that there was sufficient endogenous Sar1 in the Sar1B:H79G cell lines (Fig. 2C) to support the ER export of both Srebp2 and ATF6 precursors.

Bottom Line: The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi.The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation.We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo.

View Article: PubMed Central - PubMed

Affiliation: From the Endocrinology Centre, William Harvey Research Institute, Queen Mary University of London and Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom.

ABSTRACT
Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

Show MeSH
Related in: MedlinePlus