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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.


Loss of sgk-1 activity induced the UPR-ER.(A) Fluorescent images of worms carrying a GFP transgene under the hsp-4 promoter and treated with control RNAi (empty vector pL4440) or sgk-1 RNAi. Scale bars: 50 μm. (B) Quantitation of average pixel intensity of the images shown in (A) and 18 additional images, nine for control RNAi and nine for the sgk-1 RNAi. The average intensity is denoted with a red line. There were fifteen L4 worms in each image and ten images for each treatment. *** P value <0.001 (Student’s t-test). (C) Relative mRNA levels of hsp-4 in wild type and sgk-1(ok538) mutant animals. ** P value <0.01, * P value <0.05 (Student’s t-test).
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pone.0130778.g008: Loss of sgk-1 activity induced the UPR-ER.(A) Fluorescent images of worms carrying a GFP transgene under the hsp-4 promoter and treated with control RNAi (empty vector pL4440) or sgk-1 RNAi. Scale bars: 50 μm. (B) Quantitation of average pixel intensity of the images shown in (A) and 18 additional images, nine for control RNAi and nine for the sgk-1 RNAi. The average intensity is denoted with a red line. There were fifteen L4 worms in each image and ten images for each treatment. *** P value <0.001 (Student’s t-test). (C) Relative mRNA levels of hsp-4 in wild type and sgk-1(ok538) mutant animals. ** P value <0.01, * P value <0.05 (Student’s t-test).

Mentions: So far we provided evidence that plasma membrane proteins are mis-localized in sgk-1 mutants. One explanation for the phenotype is that the proteins do not reach the plasma membrane efficiently. Therefore, we asked next, whether the morphology of the organelles along the secretory pathway were affected in sgk-1 mutant animals. First, we examined early endosomes by checking the distribution of GFP::RAB-5 in the wild type and sgk-1(ok538) backgrounds (Fig 7, panels A and B). No obvious difference was detected, suggesting that SGK-1 function is not required for the formation of early endosomes. In earlier experiments we observed large aggregates in the sgk-1(ok538) mutant that were positive for RFP::RAB-5 and hTAC::GFP (S9 Fig), but it was unclear whether these were protein aggregates or abnormal early endosomes. Given that sgk-1(ok538) had no effect on GFP::RAB-5, they were most likely protein aggregates that contained both hTAC::GFP and RFP::RAB-5. Alternatively, loss of sgk-1 may affect early endosomes only in a sensitized background such as when the trafficking system is overloaded with cargoes. Similarly, loss of sgk-1 did not change the morphology of the Golgi compartment labeled by mCHERRY::MANS (mannosidase), which appeared as dispersed punctate structures in the intestinal cells (Fig 7, panels C and D). Next, we examined the morphology of ER using a GFP-tagged TRAM (translocating chain-associating membrane protein), an ER-specific marker, and found no obvious difference between wild type and the sgk-1 mutant (Fig 7, panels E and F). However, loss of sgk-1 induced unfolded protein response in the ER (UPR-ER) as indicated by an elevated expression of hsp-4pr::GFP (Fig 8, panels A and B). We confirmed the UPR induction by detecting increased hsp-4 mRNA levels in sgk-1(ok538) L4 mutant worms (Fig 8C). Together, these results indicate that the morphology of early endosomes, Golgi and ER is not grossly altered and that sgk-1 may be important for protein 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)

Loss of sgk-1 activity induced the UPR-ER.(A) Fluorescent images of worms carrying a GFP transgene under the hsp-4 promoter and treated with control RNAi (empty vector pL4440) or sgk-1 RNAi. Scale bars: 50 μm. (B) Quantitation of average pixel intensity of the images shown in (A) and 18 additional images, nine for control RNAi and nine for the sgk-1 RNAi. The average intensity is denoted with a red line. There were fifteen L4 worms in each image and ten images for each treatment. *** P value <0.001 (Student’s t-test). (C) Relative mRNA levels of hsp-4 in wild type and sgk-1(ok538) mutant animals. ** P value <0.01, * P value <0.05 (Student’s t-test).
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Related In: Results  -  Collection

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pone.0130778.g008: Loss of sgk-1 activity induced the UPR-ER.(A) Fluorescent images of worms carrying a GFP transgene under the hsp-4 promoter and treated with control RNAi (empty vector pL4440) or sgk-1 RNAi. Scale bars: 50 μm. (B) Quantitation of average pixel intensity of the images shown in (A) and 18 additional images, nine for control RNAi and nine for the sgk-1 RNAi. The average intensity is denoted with a red line. There were fifteen L4 worms in each image and ten images for each treatment. *** P value <0.001 (Student’s t-test). (C) Relative mRNA levels of hsp-4 in wild type and sgk-1(ok538) mutant animals. ** P value <0.01, * P value <0.05 (Student’s t-test).
Mentions: So far we provided evidence that plasma membrane proteins are mis-localized in sgk-1 mutants. One explanation for the phenotype is that the proteins do not reach the plasma membrane efficiently. Therefore, we asked next, whether the morphology of the organelles along the secretory pathway were affected in sgk-1 mutant animals. First, we examined early endosomes by checking the distribution of GFP::RAB-5 in the wild type and sgk-1(ok538) backgrounds (Fig 7, panels A and B). No obvious difference was detected, suggesting that SGK-1 function is not required for the formation of early endosomes. In earlier experiments we observed large aggregates in the sgk-1(ok538) mutant that were positive for RFP::RAB-5 and hTAC::GFP (S9 Fig), but it was unclear whether these were protein aggregates or abnormal early endosomes. Given that sgk-1(ok538) had no effect on GFP::RAB-5, they were most likely protein aggregates that contained both hTAC::GFP and RFP::RAB-5. Alternatively, loss of sgk-1 may affect early endosomes only in a sensitized background such as when the trafficking system is overloaded with cargoes. Similarly, loss of sgk-1 did not change the morphology of the Golgi compartment labeled by mCHERRY::MANS (mannosidase), which appeared as dispersed punctate structures in the intestinal cells (Fig 7, panels C and D). Next, we examined the morphology of ER using a GFP-tagged TRAM (translocating chain-associating membrane protein), an ER-specific marker, and found no obvious difference between wild type and the sgk-1 mutant (Fig 7, panels E and F). However, loss of sgk-1 induced unfolded protein response in the ER (UPR-ER) as indicated by an elevated expression of hsp-4pr::GFP (Fig 8, panels A and B). We confirmed the UPR induction by detecting increased hsp-4 mRNA levels in sgk-1(ok538) L4 mutant worms (Fig 8C). Together, these results indicate that the morphology of early endosomes, Golgi and ER is not grossly altered and that sgk-1 may be important for protein 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.