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

Sylvie Demaretz,Dalal Bakhos-Douaihy,Kamel Laghmani,Nadia Frachon,Elie Seaayfan,Martin Kömhoff

doi:10.3390/cells11010101

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.

Highlights

The sodium balance and its regulation by the kidney, by regulating the extra-cellular volume, is a key determinant of long-term blood pressure (BP) control, as illustrated by rare monogenic syndromes affecting renal salt handling and considerably altering BP [1,2]. several factors contribute to the pathogenesis and maintenance of blood pressure elevation, renal mechanisms are believed to play a primary role, as hypothesized initially by Guyton [1]
To check whether full-length NKCC2 might interact with mannosidase IA (ManIA) in a cellular context, Myc-NKCC2 construct was transiently expressed in the presence or absence of ManIA-V5 in Human embryonic kidney (HEK) cells
The association appears to be specific since Myc-NKCC2 protein was not recovered in control experiments (Figure 1B, lane 2) in which cells were transfected in the absence of ManIA-V5

Summary

Introduction

The sodium balance and its regulation by the kidney, by regulating the extra-cellular volume, is a key determinant of long-term blood pressure (BP) control, as illustrated by rare monogenic syndromes affecting renal salt handling and considerably altering BP [1,2]. several factors contribute to the pathogenesis and maintenance of blood pressure elevation, renal mechanisms are believed to play a primary role, as hypothesized initially by Guyton [1]. The sodium balance and its regulation by the kidney, by regulating the extra-cellular volume, is a key determinant of long-term blood pressure (BP) control, as illustrated by rare monogenic syndromes affecting renal salt handling and considerably altering BP [1,2]. TAL Na-Cl reabsorption is mediated by luminal Na-K-2Cl cotransporter NKCC2 [5]. NKCC2 transport function has a considerable impact on final urinary salt excretion, subsequently influencing long-term blood sodium balance [3,5]. Inherited variations of the cotransporter and/or its regulators affect BP in humans [3,6,7,8]

Methods

Results

Conclusion