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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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Sar1A and Sar1B have opposite effects on apoB48- and apoB100-containing lipoprotein secretion.A and B, McArdle-RH7777 cells were labeled with l-[35S]methionine for 60 min and chased for 120 min. The l-35S-labeled apoB in the cell media was immunoprecipitated, analyzed by SDS-PAGE, and visualized by phosphorimaging. Data (mean ± S.E.) are from four independent experiments.****, p < 0.0001 versus cells with empty vector control. ***, p < 0.001 versus cells with empty vector control. **, p < 0.01 versus cells with empty vector control. C and D, densities of fractions containing secreted l-35S-labeled apoB48-containing (C) and l-35S-labeled apoB100-containing (D) lipoproteins were determined gravimetrically. Data (mean ± S.E.) are from three independent experiments. E, secreted l-35S-labeled apoB from two independent Sar1A-FLAG cell lines, quantified as in A and B. ****, p < 0.0001 versus cells with empty vector control. **, p < 0.01 versus apoB100 lipoproteins. F, total l-[35S]methionine in secreted protein fraction. Data (mean ± S.E.) are from five independent experiments. ****, p < 0.0001 versus cells with empty vector control.
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Figure 1: Sar1A and Sar1B have opposite effects on apoB48- and apoB100-containing lipoprotein secretion.A and B, McArdle-RH7777 cells were labeled with l-[35S]methionine for 60 min and chased for 120 min. The l-35S-labeled apoB in the cell media was immunoprecipitated, analyzed by SDS-PAGE, and visualized by phosphorimaging. Data (mean ± S.E.) are from four independent experiments.****, p < 0.0001 versus cells with empty vector control. ***, p < 0.001 versus cells with empty vector control. **, p < 0.01 versus cells with empty vector control. C and D, densities of fractions containing secreted l-35S-labeled apoB48-containing (C) and l-35S-labeled apoB100-containing (D) lipoproteins were determined gravimetrically. Data (mean ± S.E.) are from three independent experiments. E, secreted l-35S-labeled apoB from two independent Sar1A-FLAG cell lines, quantified as in A and B. ****, p < 0.0001 versus cells with empty vector control. **, p < 0.01 versus apoB100 lipoproteins. F, total l-[35S]methionine in secreted protein fraction. Data (mean ± S.E.) are from five independent experiments. ****, p < 0.0001 versus cells with empty vector control.

Mentions: Sar1B:H79G overexpression virtually abolished the secretion of both apoB48 and apoB100 lipoproteins (Fig. 1A). By contrast, Sar1B overexpression increased their secretion by >3-fold (Fig. 1B). Their densities were not decreased (Fig. 1, C and D) with both the apoB48- and apoB100-containing lipoproteins exhibiting their expected densities (13). Sar1A overexpression markedly reduced apoB lipoprotein secretion (Fig. 1E); however, the effect was more pronounced for apoB100 than apoB48 (secretion 3.49 ± 1.5% (apoB100) and 20.65 ± 3.97% (apoB48) of control cells, respectively; p = 0.0016 for difference). Total protein secretion was also decreased in the Sar1B:H79G cell lines (Fig. 1F), but this reduction was significantly less than that observed for apoB48/100 (Fig. 1A). Collectively, these data show that Sar1B promotes secretion of both apoB48 and apoB100 lipoproteins, whereas Sar1A overexpression has the opposite effect, preferentially blocking the secretion of the more lipid-laden particles.


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)

Sar1A and Sar1B have opposite effects on apoB48- and apoB100-containing lipoprotein secretion.A and B, McArdle-RH7777 cells were labeled with l-[35S]methionine for 60 min and chased for 120 min. The l-35S-labeled apoB in the cell media was immunoprecipitated, analyzed by SDS-PAGE, and visualized by phosphorimaging. Data (mean ± S.E.) are from four independent experiments.****, p < 0.0001 versus cells with empty vector control. ***, p < 0.001 versus cells with empty vector control. **, p < 0.01 versus cells with empty vector control. C and D, densities of fractions containing secreted l-35S-labeled apoB48-containing (C) and l-35S-labeled apoB100-containing (D) lipoproteins were determined gravimetrically. Data (mean ± S.E.) are from three independent experiments. E, secreted l-35S-labeled apoB from two independent Sar1A-FLAG cell lines, quantified as in A and B. ****, p < 0.0001 versus cells with empty vector control. **, p < 0.01 versus apoB100 lipoproteins. F, total l-[35S]methionine in secreted protein fraction. Data (mean ± S.E.) are from five independent experiments. ****, p < 0.0001 versus cells with empty vector control.
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Figure 1: Sar1A and Sar1B have opposite effects on apoB48- and apoB100-containing lipoprotein secretion.A and B, McArdle-RH7777 cells were labeled with l-[35S]methionine for 60 min and chased for 120 min. The l-35S-labeled apoB in the cell media was immunoprecipitated, analyzed by SDS-PAGE, and visualized by phosphorimaging. Data (mean ± S.E.) are from four independent experiments.****, p < 0.0001 versus cells with empty vector control. ***, p < 0.001 versus cells with empty vector control. **, p < 0.01 versus cells with empty vector control. C and D, densities of fractions containing secreted l-35S-labeled apoB48-containing (C) and l-35S-labeled apoB100-containing (D) lipoproteins were determined gravimetrically. Data (mean ± S.E.) are from three independent experiments. E, secreted l-35S-labeled apoB from two independent Sar1A-FLAG cell lines, quantified as in A and B. ****, p < 0.0001 versus cells with empty vector control. **, p < 0.01 versus apoB100 lipoproteins. F, total l-[35S]methionine in secreted protein fraction. Data (mean ± S.E.) are from five independent experiments. ****, p < 0.0001 versus cells with empty vector control.
Mentions: Sar1B:H79G overexpression virtually abolished the secretion of both apoB48 and apoB100 lipoproteins (Fig. 1A). By contrast, Sar1B overexpression increased their secretion by >3-fold (Fig. 1B). Their densities were not decreased (Fig. 1, C and D) with both the apoB48- and apoB100-containing lipoproteins exhibiting their expected densities (13). Sar1A overexpression markedly reduced apoB lipoprotein secretion (Fig. 1E); however, the effect was more pronounced for apoB100 than apoB48 (secretion 3.49 ± 1.5% (apoB100) and 20.65 ± 3.97% (apoB48) of control cells, respectively; p = 0.0016 for difference). Total protein secretion was also decreased in the Sar1B:H79G cell lines (Fig. 1F), but this reduction was significantly less than that observed for apoB48/100 (Fig. 1A). Collectively, these data show that Sar1B promotes secretion of both apoB48 and apoB100 lipoproteins, whereas Sar1A overexpression has the opposite effect, preferentially blocking the secretion of the more lipid-laden particles.

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