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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.

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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 alters Golgi membrane organization. (A) HeLa cells expressing mannosidase II–GFP (MannII-GFP) were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of the Golgi markers mannosidase II–GFP and GRASP65 was monitored by immunofluorescence microscopy. (B) HeLa cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. (C) HeLa cells were transfected with sialyltransferase-GFP (ST-GFP) and treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of ST-GFP and TGN46 was monitored by immunofluorescence microscopy. (D) Quantitation of the relative colocalization of the different proteins in the experiments shown in A–C, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. Bars, 5 µm. **, P < 0.01.
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fig1: d-cer-C6 treatment alters Golgi membrane organization. (A) HeLa cells expressing mannosidase II–GFP (MannII-GFP) were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of the Golgi markers mannosidase II–GFP and GRASP65 was monitored by immunofluorescence microscopy. (B) HeLa cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. (C) HeLa cells were transfected with sialyltransferase-GFP (ST-GFP) and treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of ST-GFP and TGN46 was monitored by immunofluorescence microscopy. (D) Quantitation of the relative colocalization of the different proteins in the experiments shown in A–C, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. Bars, 5 µm. **, P < 0.01.

Mentions: As reported previously, perturbation of SM levels by treating cells with 20 µM d-cer-C6 blocks transport carrier biogenesis and protein transport at the Golgi complex (Duran et al., 2012). To test whether SM organization also plays a role in the organization of Golgi proteins, HeLa cells expressing the Golgi marker mannosidase II–GFP were treated for 4 h with d-cer-C6, its nonmetabolizable stereoisomer l-ceramide-C6 (l-cer-C6), or carrier as a control, and the localization of mannosidase II–GFP and the Golgi protein GRASP65 (Barr et al., 1998) was investigated by immunofluorescence microscopy. In control and l-cer-C6–treated cells, mannosidase II–GFP and GRASP65 colocalize in the perinuclear area (Fig. 1 A); however, in d-cer-C6–treated cells, these proteins are separated from each other (Fig. 1, A and D). Under the same experimental conditions, we investigated the localization of p230 and TGN46, two proteins of the TGN. In both control and l-cer-C6–treated cells, these two proteins show a high degree of colocalization, whereas in d-cer-C6–treated cells, the distribution of these two proteins is disrupted (Fig. 1, B and D). Similar results were obtained when the localization of the TGN marker ST-GFP and TGN46 was investigated (Fig. 1, C and D). It was previously shown that complex sphingolipid biosynthesis is required for the retention of a Golgi-resident mannosyltransferase in yeast (Wood et al., 2012). Our findings show that the localization of Golgi-specific proteins to the respective cisternae is perturbed upon affecting the levels of SM in mammalian cells.


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 alters Golgi membrane organization. (A) HeLa cells expressing mannosidase II–GFP (MannII-GFP) were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of the Golgi markers mannosidase II–GFP and GRASP65 was monitored by immunofluorescence microscopy. (B) HeLa cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. (C) HeLa cells were transfected with sialyltransferase-GFP (ST-GFP) and treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of ST-GFP and TGN46 was monitored by immunofluorescence microscopy. (D) Quantitation of the relative colocalization of the different proteins in the experiments shown in A–C, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. Bars, 5 µm. **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151138&req=5

fig1: d-cer-C6 treatment alters Golgi membrane organization. (A) HeLa cells expressing mannosidase II–GFP (MannII-GFP) were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of the Golgi markers mannosidase II–GFP and GRASP65 was monitored by immunofluorescence microscopy. (B) HeLa cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. (C) HeLa cells were transfected with sialyltransferase-GFP (ST-GFP) and treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The localization of ST-GFP and TGN46 was monitored by immunofluorescence microscopy. (D) Quantitation of the relative colocalization of the different proteins in the experiments shown in A–C, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. Bars, 5 µm. **, P < 0.01.
Mentions: As reported previously, perturbation of SM levels by treating cells with 20 µM d-cer-C6 blocks transport carrier biogenesis and protein transport at the Golgi complex (Duran et al., 2012). To test whether SM organization also plays a role in the organization of Golgi proteins, HeLa cells expressing the Golgi marker mannosidase II–GFP were treated for 4 h with d-cer-C6, its nonmetabolizable stereoisomer l-ceramide-C6 (l-cer-C6), or carrier as a control, and the localization of mannosidase II–GFP and the Golgi protein GRASP65 (Barr et al., 1998) was investigated by immunofluorescence microscopy. In control and l-cer-C6–treated cells, mannosidase II–GFP and GRASP65 colocalize in the perinuclear area (Fig. 1 A); however, in d-cer-C6–treated cells, these proteins are separated from each other (Fig. 1, A and D). Under the same experimental conditions, we investigated the localization of p230 and TGN46, two proteins of the TGN. In both control and l-cer-C6–treated cells, these two proteins show a high degree of colocalization, whereas in d-cer-C6–treated cells, the distribution of these two proteins is disrupted (Fig. 1, B and D). Similar results were obtained when the localization of the TGN marker ST-GFP and TGN46 was investigated (Fig. 1, C and D). It was previously shown that complex sphingolipid biosynthesis is required for the retention of a Golgi-resident mannosyltransferase in yeast (Wood et al., 2012). Our findings show that the localization of Golgi-specific proteins to the respective cisternae is perturbed upon affecting the levels of SM in mammalian cells.

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