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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.


Defective membrane trafficking of hTAC::GFP and GLUT1::GFP in sgk-1() mutants.Confocal images of wild type (A, A’, G), sgk-1(ok538) (B, B’, H) and sgk-1(mg455) (C, C’) intestinal cells expressing hTAC::GFP or GLUT1::GFP. The apical, basal and lateral membranes are indicated by open arrow heads, solid arrow heads and arrows, respectively. Scale bars: 5 μm. See Fig 2 legend for explanations of different focal planes. (D-F) Quantitation of average pixel intensity of hTAC::GFP localized in the cytoplasm (D), on the basal membrane (E) or lateral membrane (F). The average intensity is denoted with a red line. *** P value <0.001, ** P value <0.01 (Wilcoxon rank sum test).
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pone.0130778.g003: Defective membrane trafficking of hTAC::GFP and GLUT1::GFP in sgk-1() mutants.Confocal images of wild type (A, A’, G), sgk-1(ok538) (B, B’, H) and sgk-1(mg455) (C, C’) intestinal cells expressing hTAC::GFP or GLUT1::GFP. The apical, basal and lateral membranes are indicated by open arrow heads, solid arrow heads and arrows, respectively. Scale bars: 5 μm. See Fig 2 legend for explanations of different focal planes. (D-F) Quantitation of average pixel intensity of hTAC::GFP localized in the cytoplasm (D), on the basal membrane (E) or lateral membrane (F). The average intensity is denoted with a red line. *** P value <0.001, ** P value <0.01 (Wilcoxon rank sum test).

Mentions: Given that we only investigated a cargo that recycles through the Golgi so far, we asked next whether recycling to the plasma membrane was generally affected in sgk-1 mutant animals. hTAC (the a-chain of the human IL-2 receptor TAC) and GLUT1 (glucose transporter 1) are plasma membrane receptors internalized via clathrin-independent endocytosis and recycled back to the plasma membrane through the endocytic recycling compartment (ERC) and RME-1-positive basolateral recycling endosomes [44, 45]. hTAC::GFP mainly localized to the basolateral membrane in the wild type (Fig 3, panels A and A’). In the sgk-1(ok538) mutant, the pool of hTAC::GFP on the basolateral membrane decreased slightly. However, strikingly, large hTAC::GFP structures, possibly aggregated vesicles, accumulated in the cytoplasm (Fig 3, panels B, B’ and D). In the sgk-1(mg455) mutant, similar cytoplasmic accumulation of hTAC::GFP was observed, as well as a slight reduction of hTAC::GFP on the basal membrane (Fig 3, panels C, C’, D, E and F, and S1, S2 and S6–S8 Figs). To further explore whether the internal aggregations are abnormal early endosomes, we determined the distribution of hTAC::GFP and an early endosome marker, RFP::RAB-5 in wild type and sgk-1(ok538) mutant worms. In wild type worms, only a small portion of the cytoplasmic hTAC::GFP puncta co-localized with early endosomes labeled by RFP::RAB-5 (S9 Fig) In sgk-1(ok538) mutant worms, most of the hTAC::GFP labeled large, aggregated structures that are also labeled by RFP::RAB-5 (S9 Fig). It is unclear whether these structures were abnormal early endosomes or protein aggregates that contain both hTAC::GFP and RFP::RAB-5.


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)

Defective membrane trafficking of hTAC::GFP and GLUT1::GFP in sgk-1() mutants.Confocal images of wild type (A, A’, G), sgk-1(ok538) (B, B’, H) and sgk-1(mg455) (C, C’) intestinal cells expressing hTAC::GFP or GLUT1::GFP. The apical, basal and lateral membranes are indicated by open arrow heads, solid arrow heads and arrows, respectively. Scale bars: 5 μm. See Fig 2 legend for explanations of different focal planes. (D-F) Quantitation of average pixel intensity of hTAC::GFP localized in the cytoplasm (D), on the basal membrane (E) or lateral membrane (F). The average intensity is denoted with a red line. *** P value <0.001, ** P value <0.01 (Wilcoxon rank sum test).
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Related In: Results  -  Collection

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pone.0130778.g003: Defective membrane trafficking of hTAC::GFP and GLUT1::GFP in sgk-1() mutants.Confocal images of wild type (A, A’, G), sgk-1(ok538) (B, B’, H) and sgk-1(mg455) (C, C’) intestinal cells expressing hTAC::GFP or GLUT1::GFP. The apical, basal and lateral membranes are indicated by open arrow heads, solid arrow heads and arrows, respectively. Scale bars: 5 μm. See Fig 2 legend for explanations of different focal planes. (D-F) Quantitation of average pixel intensity of hTAC::GFP localized in the cytoplasm (D), on the basal membrane (E) or lateral membrane (F). The average intensity is denoted with a red line. *** P value <0.001, ** P value <0.01 (Wilcoxon rank sum test).
Mentions: Given that we only investigated a cargo that recycles through the Golgi so far, we asked next whether recycling to the plasma membrane was generally affected in sgk-1 mutant animals. hTAC (the a-chain of the human IL-2 receptor TAC) and GLUT1 (glucose transporter 1) are plasma membrane receptors internalized via clathrin-independent endocytosis and recycled back to the plasma membrane through the endocytic recycling compartment (ERC) and RME-1-positive basolateral recycling endosomes [44, 45]. hTAC::GFP mainly localized to the basolateral membrane in the wild type (Fig 3, panels A and A’). In the sgk-1(ok538) mutant, the pool of hTAC::GFP on the basolateral membrane decreased slightly. However, strikingly, large hTAC::GFP structures, possibly aggregated vesicles, accumulated in the cytoplasm (Fig 3, panels B, B’ and D). In the sgk-1(mg455) mutant, similar cytoplasmic accumulation of hTAC::GFP was observed, as well as a slight reduction of hTAC::GFP on the basal membrane (Fig 3, panels C, C’, D, E and F, and S1, S2 and S6–S8 Figs). To further explore whether the internal aggregations are abnormal early endosomes, we determined the distribution of hTAC::GFP and an early endosome marker, RFP::RAB-5 in wild type and sgk-1(ok538) mutant worms. In wild type worms, only a small portion of the cytoplasmic hTAC::GFP puncta co-localized with early endosomes labeled by RFP::RAB-5 (S9 Fig) In sgk-1(ok538) mutant worms, most of the hTAC::GFP labeled large, aggregated structures that are also labeled by RFP::RAB-5 (S9 Fig). It is unclear whether these structures were abnormal early endosomes or protein aggregates that contain both hTAC::GFP and RFP::RAB-5.

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.