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. 2011 Dec 14:8:89.
doi: 10.1186/1743-7075-8-89.

Subcellular targeting domains of sphingomyelin synthase 1 and 2

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Subcellular targeting domains of sphingomyelin synthase 1 and 2

Calvin Yeang et al. Nutr Metab (Lond). .

Abstract

Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

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Figures

Figure 1
Figure 1
Localization of GFP-SMS1 and 2 fusion proteins. Confocal microscopy showing co-localization of TGN46 (Golgi marker) and Wheat Germ Agglutinin (WGA, plasma membrane marker) with GFP-SMS1 (A) and GFP-SMS2 (B). Schematics depict predicted transmembrane (TM) domains in SMS1 and SMS2. TOPRO is a marker for nucleus.
Figure 2
Figure 2
BFA blocks sorting of SMS1 and 2. HeLa cells expressing GFP-SMS1 (A and D), GFP-SMS2 (B and E), CD4-GFP (C and F) were treated with BFA (5 μg/ml) for 8 hours (A-C). The cells were washed 6 times with growth media to remove BFA and allowed to recover for 3 h (D-F). Images were obtained using confocal microscopy. Calnexin is an ER marker, TGN46 is a Golgi marker, and Wheat Germ Agglutinin (WGA) is a plasma membrane marker.
Figure 3
Figure 3
Localization of N-terminal truncation mutants of SMS1 and SMS2. HeLa cells expressing GFP-SMS1 N130 (A) and GFP-SMS2 N60 (B). The organelle markers used were calnexin (ER) and TOPRO 3 (nucleus). The images were obtained using confocal microscopy.
Figure 4
Figure 4
Localization of SMS1 and SMS2 N-terminal chimeras. HeLa cells transfected with GFP-nS2/S1 (A) or GFP-nS1/S2 (B). The organelle markers were TGN46 (trans-Golgi) and WGA (plasma membrane). Images were obtained using confocal microscopy.
Figure 5
Figure 5
Localization of SMS1 and SMS2 C-terminal chimeras and mutants. Confocal images of HeLa cells transfected with A) GFP-S1/cS2, B) GFP-S2/cS1, C) GFP-S2ΔC32, D) GFP-S2ΔC67, E) GFP-S1ΔC4, F) GFP-S1ΔC17, and G) GFP-S1ΔC27. Organelle markers used were TGN46 (Golgi) and WGA (plasma membrane).

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