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Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2

Abstract : Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.
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Submitted on : Friday, January 14, 2022 - 11:30:27 AM
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Sylvie Demaretz, Elie Seaayfan, Dalal Bakhos-Douaihy, Nadia Frachon, Martin Kömhoff, et al.. Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2. Cells, MDPI, 2021, 11 (1), pp.101. ⟨10.3390/cells11010101⟩. ⟨hal-03526059⟩

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