AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2

Abstract

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

Protein Quality Control of NKCC2 in Bartter Syndrome and Blood Pressure Regulation

“…Given AUP1's hairpin topology in the ER membrane, with the N and C tails of the protein facing the cytosol [59], and that OS9 might be present at both the luminal and cytoplasmic sides of the ER [55,60], an interaction between these proteins and NKCC2 at the cytoplasmic surface of the ER is conceivable. Using two different kidney cell lines (HEK and OKP cells), we provided evidence that both OS9 and AUP1 interact with the ER-resident form of NKCC2, which is coherent with a potential role in the ERAD of the cotransporter [44,45]. Accordingly, OS9 and AUP1 overexpression decreased the stability and the maturation of NKCC2, whereas their knockdown produced the opposite effect.…”

“…Mutating NKCC2 N-glycosylation sites or inhibiting mannose-trimming with kifunensine completely abolished the effects of OS9 and AUP1 on NKCC2, indicating that the enhanced degradation of the cotransporter is N-glycan-dependent. Importantly, AUP1 and OS9 influences on immature NKCC2 were also abrogated by MG132, suggesting that OS9 and AUP1 target the core glycosylated form of NKCC2 to the proteasome-dependent ERAD pathway [44,45]. Furthermore, in the presence of MG132, the increase in NKCC2 protein amount upon the inhibition of endogenous AUP1 was associated with a striking decrease in polyubiquitinated NKCC2 species, demonstrating that AUP1 enhances cotransporter ERAD by promoting its polyubiquitination [44].…”

“…Importantly, AUP1 and OS9 influences on immature NKCC2 were also abrogated by MG132, suggesting that OS9 and AUP1 target the core glycosylated form of NKCC2 to the proteasome-dependent ERAD pathway [44,45]. Furthermore, in the presence of MG132, the increase in NKCC2 protein amount upon the inhibition of endogenous AUP1 was associated with a striking decrease in polyubiquitinated NKCC2 species, demonstrating that AUP1 enhances cotransporter ERAD by promoting its polyubiquitination [44]. Notably, although the effects of AUP1 and OS9 on NKCC2 were similar, they were not additive, strongly suggesting that both proteins work on the same molecular pathway to regulate the ERAD of NKCC2.…”

“…While STCH and ManIA bind to the proximal region of the NKCC2 C-terminal tail [42,43], AUP1 interacts with the intermediate region [44]. OS9 binds to both the intermediate [44] and distal regions of the NKCC2 carboxy-terminal tail [45]. To identify the ER export signals of NKCC2, we focused on the region downstream from the Y998X mutant that harbors several di-leucine-like motifs and consensus di-acidic (D/E-X-D/E) codes.…”

“…Using Y2H, the ER-lectin OS9 (Osteosarcoma amplified 9) and AUP1 (Ancient Ubiquitous Protein 1) were identified as novel interactors in the NKCC2 COOH terminus (Figure 1) [44,45]. This is of great importance because interaction between AUP1 and OS9 has been reported [54], opening, therefore, the possibility for a tripartite complex between these two proteins and NKCC2.…”