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Abstract: PO1403

STCH Regulates NKCC2 Biogenesis by Both the Endoplasmic Reticulum-Associated Degradation and the Endoplasmic Reticulum-to-Lysosome-Associated Degradation Pathways

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Bakhos Al Douaihy, Dalal, Sorbonne Universite, Paris, Île-de-France, France
  • Seaayfan, Elie, Philipps Univ Marburgersity, Marburg, Germany
  • Demaretz, Sylvie, Sorbonne Universite, Paris, Île-de-France, France
  • Hamdani, Mehdi, Sorbonne Universite, Paris, Île-de-France, France
  • Kömhoff, Martin, Philipps Univ Marburgersity, Marburg, Germany
  • Laghmani, Kamel, Sorbonne Universite, Paris, Île-de-France, France

Group or Team Name

  • Team 3
Background

Mutations in the apically located Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in the maturation and cell surface expression of NKCC2. Yet very little is known about the molecular components of NKCC2 ER quality control. Using the yeast two hybrid system and co-immunoprecipitation assays, we identified chaperone stress 70 protein (STCH), as a binding partner of the immature form of NKCC2. STCH is supposed to function as an ER chaperone but the precise molecular role of STCH remains obscure.

Methods

Protein expression was monitored in transiently transfected HEK cells, using immunoblot and confocal imaging. Protein maturation and stability were assessed by Endo-H digestion and cycloheximide chase (CHX) assay.

Results

Co-immunolocalization experiments revealed that NKCC2 interacts with STCH mainly at the ER. However, CHX assay together with Endo-H digestion revealed that STCH is initially synthesized in the ER as a core-glycosylated protein before being gradually converted to a hybrid N-glycosylated form. These data are in an agreement with a previous study showing that STCH contains a mannose-6-phosphorylation site, suggesting therefore that STCH expression is not restricted to the ER. STCH knock-down increased NKCC2 protein abundance in a dose-depend manner, whereas STCH over-expression had the opposite effect. CHX assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. STCH induced reduction in NKCC2 expression were offset partially by the proteasome inhibitor MG132. Interestingly, leupeptin and chloroquine, two potent inhibitors of the lysosome, mimicked MG132 effect on NKCC2 regulation. Accordingly, the simultaneous presence of proteasome and lysosome inhibitors, completely abolished STCH-induced down-regulation of NKCC2.

Conclusion

Our data demonstrate the presence of an STCH mediated ER quality control of NKCC2 in renal cells. They suggest a model whereby, in addition to the proteasome-dependent ERAD, the ER quality control of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway, revealing therefore a new regulatory mechanism governing the co-transporter biogenesis.

Funding

  • Government Support - Non-U.S.