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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–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, 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. **, P < 0.01.
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fig3: d-cer-C6–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, 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. **, P < 0.01.

Mentions: We reported previously that upon treatment of cells with d-cer-C6, C6-SM is generated by SMS1 and SMS2, which replaces endogenous SM and ultimately leads to inhibition of protein exit from the Golgi complex (Duran et al., 2012). To test whether formation of C6-SM from d-cer-C6 plays a role in the observed effects in Golgi compartmentalization (Fig. 1), SMS1 and SMS2 were knocked down in HeLa cells by siRNA. Measurement of the knockdown efficiency by RT-PCR showed a reduction in SMS1 and SMS2 mRNA levels by 65 and 50%, respectively, compared with control siRNA–transfected cells (Fig. 3 A). HeLa cells transfected with control or SMS1 and SMS2 siRNA were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the two TGN proteins p230 and TGN46 was assessed by immunofluorescence microscopy. In control siRNA–transfected cells, after d-cer-C6 treatment, we observed an altered TGN morphology and segregation of both proteins within the TGN as compared with both ethanol- or l-cer-C6–treated cells (Fig. 3, B [top] and C). However, the change in the location of these proteins with respect to each other upon d-cer-C6 treatment was inhibited in cells in which SMS1 and SMS2 were knocked down (Fig. 3, B [bottom] and C). Collectively, these results indicate that the observed effects on the compartmentalization and morphology of the different Golgi cisternae after d-cer-C6 treatment are caused by the conversion of this lipid to C6-SM. In addition, our previously reported results on the lipid profile of Golgi membranes from cells treated with d-cer-C6 showed that the formation of the C6-SM occurred at the expense of endogenous long-chain SM, whereas the total levels of SM remained constant (Duran et al., 2012). Our results hence show the importance of regulated levels of SM for the lateral organization of Golgi proteins.


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–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, 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. **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4151138&req=5

fig3: d-cer-C6–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, 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. **, P < 0.01.
Mentions: We reported previously that upon treatment of cells with d-cer-C6, C6-SM is generated by SMS1 and SMS2, which replaces endogenous SM and ultimately leads to inhibition of protein exit from the Golgi complex (Duran et al., 2012). To test whether formation of C6-SM from d-cer-C6 plays a role in the observed effects in Golgi compartmentalization (Fig. 1), SMS1 and SMS2 were knocked down in HeLa cells by siRNA. Measurement of the knockdown efficiency by RT-PCR showed a reduction in SMS1 and SMS2 mRNA levels by 65 and 50%, respectively, compared with control siRNA–transfected cells (Fig. 3 A). HeLa cells transfected with control or SMS1 and SMS2 siRNA were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h, and the localization of the two TGN proteins p230 and TGN46 was assessed by immunofluorescence microscopy. In control siRNA–transfected cells, after d-cer-C6 treatment, we observed an altered TGN morphology and segregation of both proteins within the TGN as compared with both ethanol- or l-cer-C6–treated cells (Fig. 3, B [top] and C). However, the change in the location of these proteins with respect to each other upon d-cer-C6 treatment was inhibited in cells in which SMS1 and SMS2 were knocked down (Fig. 3, B [bottom] and C). Collectively, these results indicate that the observed effects on the compartmentalization and morphology of the different Golgi cisternae after d-cer-C6 treatment are caused by the conversion of this lipid to C6-SM. In addition, our previously reported results on the lipid profile of Golgi membranes from cells treated with d-cer-C6 showed that the formation of the C6-SM occurred at the expense of endogenous long-chain SM, whereas the total levels of SM remained constant (Duran et al., 2012). Our results hence show the importance of regulated levels of SM for the lateral organization of Golgi proteins.

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