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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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Effect of constitutively active Sar1B and native Sar1 expression on ApoB and Mttp expression in McArdle-RH7777 cells.A and B, isoform-specific effects of Sar1a and Sar1b knockdown on transcript (A) and protein (B) analyzed by RT-qPCR and quantitative Western blot analysis. NQ, not quantified due to undetectable levels of Sar1b in some cell lysates. White line separators indicate that noncontiguous lanes from the same gel are shown. A, *, p < 0.05 versus Sar1a + Sar1b knockdown. B, ***, p < 0.001 versus scrambled control siRNA; **, p < 0.01 versus scrambled control siRNA. C, Sar1a protein in stably overexpressing Sar1B:H79G and empty vector control cells. *, p < 0.05 versus cells with empty vector control. D, Sar1 isoform-specific effects on ApoB and Mttp mRNA levels in overexpressing cell lines, analyzed by RT-qPCR. ***, p < 0.001 versus cells with empty vector control. E, Sar1b knockdown specifically reduces ApoB and Mttp mRNA, analyzed by RT-qPCR. **, p < 0.01 versus scrambled control siRNA. F and G, ApoB and Mttp knockdown (F) does not decrease Sar1a and Sar1b mRNA (G), analyzed by RT-qPCR. A–G, data (mean ± S.E.) are from at least three independent experiments.
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Figure 2: Effect of constitutively active Sar1B and native Sar1 expression on ApoB and Mttp expression in McArdle-RH7777 cells.A and B, isoform-specific effects of Sar1a and Sar1b knockdown on transcript (A) and protein (B) analyzed by RT-qPCR and quantitative Western blot analysis. NQ, not quantified due to undetectable levels of Sar1b in some cell lysates. White line separators indicate that noncontiguous lanes from the same gel are shown. A, *, p < 0.05 versus Sar1a + Sar1b knockdown. B, ***, p < 0.001 versus scrambled control siRNA; **, p < 0.01 versus scrambled control siRNA. C, Sar1a protein in stably overexpressing Sar1B:H79G and empty vector control cells. *, p < 0.05 versus cells with empty vector control. D, Sar1 isoform-specific effects on ApoB and Mttp mRNA levels in overexpressing cell lines, analyzed by RT-qPCR. ***, p < 0.001 versus cells with empty vector control. E, Sar1b knockdown specifically reduces ApoB and Mttp mRNA, analyzed by RT-qPCR. **, p < 0.01 versus scrambled control siRNA. F and G, ApoB and Mttp knockdown (F) does not decrease Sar1a and Sar1b mRNA (G), analyzed by RT-qPCR. A–G, data (mean ± S.E.) are from at least three independent experiments.

Mentions: To determine whether Sar1 also had isoform-specific effects on the expression of ApoB and Mttp, we measured the levels of their RNAs by RT-qPCR in our McArdle-RH7777 cell lines plus McArdle-RH7777 cells transfected with Sar1 siRNAs. Individual knockdown of the Sar1 isoforms were isoform-specific, and there were no increases in the mRNA (Fig. 2A) or protein (Fig. 2B) levels of the nontargeted isoform. The amounts of Sar1a protein in cells transfected with Sar1a and Sar1a plus Sar1b siRNAs were 28.16 ± 6.36 and 32.88 ± 8.70%, respectively, of that in cells transfected with the scrambled siRNA control (Fig. 2B). Sar1b knockdown also substantially reduced in Sar1b protein and in the majority of samples to undetectable levels (Fig. 2B). This was due to the Western blot analysis returning weaker signals for Sar1b than Sar1a, consistent with RT-qPCR data (Sar1b mRNA 11.6-fold lower than Sar1a mRNA), plus the efficiency of Sar1b knockdown in reducing Sar1b protein (≥70% in the rare knockdown sample where it could be quantified).


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)

Effect of constitutively active Sar1B and native Sar1 expression on ApoB and Mttp expression in McArdle-RH7777 cells.A and B, isoform-specific effects of Sar1a and Sar1b knockdown on transcript (A) and protein (B) analyzed by RT-qPCR and quantitative Western blot analysis. NQ, not quantified due to undetectable levels of Sar1b in some cell lysates. White line separators indicate that noncontiguous lanes from the same gel are shown. A, *, p < 0.05 versus Sar1a + Sar1b knockdown. B, ***, p < 0.001 versus scrambled control siRNA; **, p < 0.01 versus scrambled control siRNA. C, Sar1a protein in stably overexpressing Sar1B:H79G and empty vector control cells. *, p < 0.05 versus cells with empty vector control. D, Sar1 isoform-specific effects on ApoB and Mttp mRNA levels in overexpressing cell lines, analyzed by RT-qPCR. ***, p < 0.001 versus cells with empty vector control. E, Sar1b knockdown specifically reduces ApoB and Mttp mRNA, analyzed by RT-qPCR. **, p < 0.01 versus scrambled control siRNA. F and G, ApoB and Mttp knockdown (F) does not decrease Sar1a and Sar1b mRNA (G), analyzed by RT-qPCR. A–G, data (mean ± S.E.) are from at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of constitutively active Sar1B and native Sar1 expression on ApoB and Mttp expression in McArdle-RH7777 cells.A and B, isoform-specific effects of Sar1a and Sar1b knockdown on transcript (A) and protein (B) analyzed by RT-qPCR and quantitative Western blot analysis. NQ, not quantified due to undetectable levels of Sar1b in some cell lysates. White line separators indicate that noncontiguous lanes from the same gel are shown. A, *, p < 0.05 versus Sar1a + Sar1b knockdown. B, ***, p < 0.001 versus scrambled control siRNA; **, p < 0.01 versus scrambled control siRNA. C, Sar1a protein in stably overexpressing Sar1B:H79G and empty vector control cells. *, p < 0.05 versus cells with empty vector control. D, Sar1 isoform-specific effects on ApoB and Mttp mRNA levels in overexpressing cell lines, analyzed by RT-qPCR. ***, p < 0.001 versus cells with empty vector control. E, Sar1b knockdown specifically reduces ApoB and Mttp mRNA, analyzed by RT-qPCR. **, p < 0.01 versus scrambled control siRNA. F and G, ApoB and Mttp knockdown (F) does not decrease Sar1a and Sar1b mRNA (G), analyzed by RT-qPCR. A–G, data (mean ± S.E.) are from at least three independent experiments.
Mentions: To determine whether Sar1 also had isoform-specific effects on the expression of ApoB and Mttp, we measured the levels of their RNAs by RT-qPCR in our McArdle-RH7777 cell lines plus McArdle-RH7777 cells transfected with Sar1 siRNAs. Individual knockdown of the Sar1 isoforms were isoform-specific, and there were no increases in the mRNA (Fig. 2A) or protein (Fig. 2B) levels of the nontargeted isoform. The amounts of Sar1a protein in cells transfected with Sar1a and Sar1a plus Sar1b siRNAs were 28.16 ± 6.36 and 32.88 ± 8.70%, respectively, of that in cells transfected with the scrambled siRNA control (Fig. 2B). Sar1b knockdown also substantially reduced in Sar1b protein and in the majority of samples to undetectable levels (Fig. 2B). This was due to the Western blot analysis returning weaker signals for Sar1b than Sar1a, consistent with RT-qPCR data (Sar1b mRNA 11.6-fold lower than Sar1a mRNA), plus the efficiency of Sar1b knockdown in reducing Sar1b protein (≥70% in the rare knockdown sample where it could be quantified).

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