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Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans.

Zhu M, Wu G, Li YX, Stevens JK, Fan CX, Spang A, Dong MQ - PLoS ONE (2015)

Bottom Line: This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP.The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree.In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China; National Institute of Biological Sciences, Beijing, Beijing, China.

ABSTRACT
The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.

No MeSH data available.


sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly.Images of wild type (A, B, C) and sgk-1(ok538) mutant animals (D, E, F) treated with control RNAi (A, D) and cgt-1/3 double RNAi from Ahringer library (B, E) and RCE1181 library (C, F). The experiments started with 5 gravid adult worms on each plate and the plates were imaged 5 days later.
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pone.0130778.g006: sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly.Images of wild type (A, B, C) and sgk-1(ok538) mutant animals (D, E, F) treated with control RNAi (A, D) and cgt-1/3 double RNAi from Ahringer library (B, E) and RCE1181 library (C, F). The experiments started with 5 gravid adult worms on each plate and the plates were imaged 5 days later.

Mentions: To find out whether sgk-1 regulates membrane trafficking through sphingolipids, we first tested Myriocin, an inhibitor of the first and the key step in sphingosine biosynthesis. We fed the wild type and sgk-1(ok538) worms with 4.2 μM, 25.2 μM and 50.4 μM of Myriocin, but did not detect any obvious growth or morphological effect in either strain. Also, we did not see any effect of Myriocin on the localization pattern of either hTAC::GFP or MIG-14::GFP in wild type animals (S11 and S12 Figs). It is plausible that the drug may not accumulate efficiently in cells to show an effect. Therefore, we knocked-down cgt-1 and cgt-3, which encode two of the three ceramide glucosyltransferases that are important for the synthesis of glucosylceramide and glycosphingolipids. Similar to the cgt-1; cgt-2; cgt-3 triple mutant, cgt-1; cgt-3 double mutant animals have reduced glucosylceramide levels and arrest at the L1 larval stage [46]. In about 50% of the wild-type worms, cgt-1/3 double RNAi reduced the expression of hTAC::GFP to an undetectable level. In the remaining 50% wild-type worms, cgt-1/3 RNAi significantly decreased the amount of small cytoplasmic hTAC::GFP puncta (Fig 4). Larger-sized hTAC::GFP puncta or aggregates were seen in the cytoplasm of intestinal cells in the sgk-1 mutant, but they were reduced in number upon cgt-1/3 RNAi (Fig 4). Therefore, cgt-1/3 RNAi alleviated the phenotype of sgk-1 with respect to hTAC::GFP. Similarly, cgt-1/3 double RNAi suppressed sgk-1 with respect to MIG-14::GFP. sgk-1 nearly abolished MIG-14::GFP on the basolateral membrane, but this was restored by cgt-1/3 double RNAi (Fig 5). It has been reported that worms treated with cgt-1/3 double RNAi arrested at the L1 stage [46], but in our hands, only some of the cgt-1/3 double RNAi animals arrested at L1, probably due to a weaker RNAi effect. The sgk-1 mutant worms develop somewhat more slowly than the wild type. Interestingly, sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly (Fig 6). Altogether, our results show that sgk-1 and cgt-1/3 have complex genetic interactions. Our data indicate that SGK-1 plays a conserved role in lipid homeostasis.


Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans.

Zhu M, Wu G, Li YX, Stevens JK, Fan CX, Spang A, Dong MQ - PLoS ONE (2015)

sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly.Images of wild type (A, B, C) and sgk-1(ok538) mutant animals (D, E, F) treated with control RNAi (A, D) and cgt-1/3 double RNAi from Ahringer library (B, E) and RCE1181 library (C, F). The experiments started with 5 gravid adult worms on each plate and the plates were imaged 5 days later.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482599&req=5

pone.0130778.g006: sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly.Images of wild type (A, B, C) and sgk-1(ok538) mutant animals (D, E, F) treated with control RNAi (A, D) and cgt-1/3 double RNAi from Ahringer library (B, E) and RCE1181 library (C, F). The experiments started with 5 gravid adult worms on each plate and the plates were imaged 5 days later.
Mentions: To find out whether sgk-1 regulates membrane trafficking through sphingolipids, we first tested Myriocin, an inhibitor of the first and the key step in sphingosine biosynthesis. We fed the wild type and sgk-1(ok538) worms with 4.2 μM, 25.2 μM and 50.4 μM of Myriocin, but did not detect any obvious growth or morphological effect in either strain. Also, we did not see any effect of Myriocin on the localization pattern of either hTAC::GFP or MIG-14::GFP in wild type animals (S11 and S12 Figs). It is plausible that the drug may not accumulate efficiently in cells to show an effect. Therefore, we knocked-down cgt-1 and cgt-3, which encode two of the three ceramide glucosyltransferases that are important for the synthesis of glucosylceramide and glycosphingolipids. Similar to the cgt-1; cgt-2; cgt-3 triple mutant, cgt-1; cgt-3 double mutant animals have reduced glucosylceramide levels and arrest at the L1 larval stage [46]. In about 50% of the wild-type worms, cgt-1/3 double RNAi reduced the expression of hTAC::GFP to an undetectable level. In the remaining 50% wild-type worms, cgt-1/3 RNAi significantly decreased the amount of small cytoplasmic hTAC::GFP puncta (Fig 4). Larger-sized hTAC::GFP puncta or aggregates were seen in the cytoplasm of intestinal cells in the sgk-1 mutant, but they were reduced in number upon cgt-1/3 RNAi (Fig 4). Therefore, cgt-1/3 RNAi alleviated the phenotype of sgk-1 with respect to hTAC::GFP. Similarly, cgt-1/3 double RNAi suppressed sgk-1 with respect to MIG-14::GFP. sgk-1 nearly abolished MIG-14::GFP on the basolateral membrane, but this was restored by cgt-1/3 double RNAi (Fig 5). It has been reported that worms treated with cgt-1/3 double RNAi arrested at the L1 stage [46], but in our hands, only some of the cgt-1/3 double RNAi animals arrested at L1, probably due to a weaker RNAi effect. The sgk-1 mutant worms develop somewhat more slowly than the wild type. Interestingly, sgk-1 mutant worms treated with cgt-1/3 double RNAi developed very slowly (Fig 6). Altogether, our results show that sgk-1 and cgt-1/3 have complex genetic interactions. Our data indicate that SGK-1 plays a conserved role in lipid homeostasis.

Bottom Line: This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP.The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree.In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China; National Institute of Biological Sciences, Beijing, Beijing, China.

ABSTRACT
The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.

No MeSH data available.