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Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.

van Galen J, Campelo F, Martínez-Alonso E, Scarpa M, Martínez-Menárguez JÁ, Malhotra V - J. Cell Biol. (2014)

Bottom Line: Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins?We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other.Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Developmental Biology Programme, Centre for Genomic Regulation, 08003 Barcelona, Spain Universitat Pompeu Fabra, 08002 Barcelona, Spain.

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d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.
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fig4: d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.

Mentions: As the localization of Golgi glycosylation enzymes is affected by treatment with d-cer-C6 (Fig. 1), we investigated whether protein glycosylation was also affected. TGN46 is a transmembrane protein that is localized to the TGN and cycles between the TGN and the plasma membrane (Banting and Ponnambalam, 1997). The core protein of TGN46 has a molecular mass of 46 kD, but as a result of various glycosylations occurring at the ER and the Golgi complex, the mature protein has an apparent molecular mass of ∼110 kD (Prescott et al., 1997). HeLa cells were treated with increasing concentrations of d-cer-C6 for 4 h, after which the apparent size of TGN46 was examined by Western blotting. Whereas in cells treated with ethanol or l-cer-C6, TGN46 mainly appears as a fully processed 110-kD band, a smaller form of ∼80 kD becomes the main form upon treatments with increasing concentrations of d-cer-C6, indicating an incomplete processing of the protein (Fig. 4 A). This 80-kD form of TGN46 corresponds to a newly synthesized pool because this band was not evident in cells treated with d-cer-C6 in the presence of cycloheximide to stop new protein synthesis (Fig. S1).


Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.

van Galen J, Campelo F, Martínez-Alonso E, Scarpa M, Martínez-Menárguez JÁ, Malhotra V - J. Cell Biol. (2014)

d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.
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fig4: d-cer-C6 treatment affects TGN46 glycosylation. (A) HeLa cells were treated with ethanol, the indicated concentrations of d-cer-C6, or 20 µM l-cer-C6 for 4 h, after which the cells were lysed, and the lysates were analyzed by Western blotting using an anti-TGN46 antibody. A 110-kD band corresponds to the fully processed, fully glycosylated TGN46, whereas smaller bands of ∼95, 80, and 75 kD correspond to immature forms of TGN46. (B) HeLa cells were treated with ethanol or 20 µM d-cer-C6 for 4 h, after which the cells were biotinylated. After isolation of biotinylated proteins, the biotinylated fractions and cell lysates were treated with neuraminidase (Neur.) or buffer alone and analyzed by Western blotting using antibodies against TGN46 and β-actin. (C) HeLa cells were treated with ethanol or with 20 µM d-cer-C6 for 4 h, after which the cells were lysed. Lysates were treated with a deglycosylation (Deglyc.) mix (second and fifth lanes), treated with neuraminidase (third and sixth lanes), or remained untreated (first and fourth lanes) and analyzed by Western blotting using an anti-TGN46 antibody. (D) HeLa cells transfected with control (CTRL) or SMS1 + SMS2 siRNA for 92 h were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, after which cells were lysed, and the lysates were analyzed by Western blotting using anti-TGN46 and anti–β-actin antibodies. (E) Quantitation of the band intensity of mature 110-kD TGN46 (in percentages of total TGN46) for the experiment in D. Bars show the mean values ± SEM of four independent experiments (n = 4). Statistical significance is indicated as **, P < 0.01 or n.s., P > 0.05.
Mentions: As the localization of Golgi glycosylation enzymes is affected by treatment with d-cer-C6 (Fig. 1), we investigated whether protein glycosylation was also affected. TGN46 is a transmembrane protein that is localized to the TGN and cycles between the TGN and the plasma membrane (Banting and Ponnambalam, 1997). The core protein of TGN46 has a molecular mass of 46 kD, but as a result of various glycosylations occurring at the ER and the Golgi complex, the mature protein has an apparent molecular mass of ∼110 kD (Prescott et al., 1997). HeLa cells were treated with increasing concentrations of d-cer-C6 for 4 h, after which the apparent size of TGN46 was examined by Western blotting. Whereas in cells treated with ethanol or l-cer-C6, TGN46 mainly appears as a fully processed 110-kD band, a smaller form of ∼80 kD becomes the main form upon treatments with increasing concentrations of d-cer-C6, indicating an incomplete processing of the protein (Fig. 4 A). This 80-kD form of TGN46 corresponds to a newly synthesized pool because this band was not evident in cells treated with d-cer-C6 in the presence of cycloheximide to stop new protein synthesis (Fig. S1).

Bottom Line: Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins?We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other.Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Developmental Biology Programme, Centre for Genomic Regulation, 08003 Barcelona, Spain Universitat Pompeu Fabra, 08002 Barcelona, Spain.

Show MeSH
Related in: MedlinePlus