Article Dans Une Revue International Journal of Molecular Sciences Année : 2022

Diacidic Motifs in the Carboxyl Terminus Are Required for ER Exit and Translocation to the Plasma Membrane of NKCC2

Résumé

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises cotransporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949 EEE 951 and 1019 DAELE 1023 , located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949 EEE 951 motif to 949 AEA 951 had no effect on NKCC2 processing, mutating 1019 DAE 1021 to 1019 AAA 1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019 DAELE 1023 to 1019 AAALA 1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019 AAALA 1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019 AAALA 1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.

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hal-03874297 , version 1 (27-11-2022)

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Dalal Bakhos-Douaihy, Elie Seaayfan, Nadia Frachon, Sylvie Demaretz, Martin Kömhoff, et al.. Diacidic Motifs in the Carboxyl Terminus Are Required for ER Exit and Translocation to the Plasma Membrane of NKCC2. International Journal of Molecular Sciences, 2022, 23 (21), pp.12761. ⟨10.3390/ijms232112761⟩. ⟨hal-03874297⟩
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