Elsevier

Experimental Cell Research

Volume 315, Issue 17, 15 October 2009, Pages 3014-3027
Experimental Cell Research

Research Article
Dual degradation mechanisms ensure disposal of NHE6 mutant protein associated with neurological disease

https://doi.org/10.1016/j.yexcr.2009.07.012Get rights and content

Abstract

Clinical features characterizing Angelman syndrome, previously shown to be caused by disruption of UBE3A, were recently also described in neurologically disabled patients with mutations in SLC9A6, which encodes the Na+/H+ exchanger NHE6. In the present work we have focused on NHE6Δ255–256, the protein product of a specific 6-bp patient deletion in SLC9A6. To resolve the molecular mechanism causing the cellular dysfunction associated with this mutant, we have characterized its intracellular behaviour in comparison to wild type NHE6. Our study demonstrates that NHE6Δ255–256 is much less stable than the wild type protein. Whereas wild type NHE6 is transported to the plasma membrane and early endosomes and remains stable, NHE6Δ255–256 is degraded via two independent pathways mediated by proteasomes and lysosomes, respectively. Depletion of NHE6 had no detectable effect on endosomal pH, but co-depletion of NHE6 and the closely related NHE9 caused enhanced acidification of early endosomes. Our results suggest that NHE6 participates in regulation of endosomal pH and provides a cellular basis for understanding the loss of NHE6 function leading to a neurological phenotype resembling Angelman syndrome.

Introduction

Angelman syndrome is a neurodevelopmental disorder characterized by symptoms such as severe developmental delay, hyperkinetic movement disorder, a happy demeanour, speech impairment and epileptic seizures [1]. The genetic basis for the disorder varies from large interstitial maternally inherited deletions of chromosome 15q11–13, to alterations in imprinting and point mutations. In spite of this, all identified mechanisms involve disturbance of the gene UBE3A. The protein product of the UBE3A gene is the E3-ubiquitin ligase E6-AP, which was first identified as a mediator of ubiquitination and degradation of p53 in association with the E6 oncoprotein of the human papilloma virus [2], [3]. Apart from the virus-mediated p53 ubiquitination by E6-AP, the role of this ligase has remained elusive. UBE3A is maternally imprinted in the brain, particularly in the hippocampus and cerebellum, and studies in mice with a maternal null mutation and Angelman syndrome-related features have revealed defects in the neuronal process of long-term potentiation (LTP) [4] and abnormal dendritic spine development [5]. Moreover, these mice have reduced activity of calcium/calmodulin-dependent kinase type 2 (CaMKII) in the brain by inhibitory phosphorylation [6], and the Angelman syndrome-like features can be rescued by introducing a mutation preventing this inhibitory phosphorylation [7].

Recently, a 6-bp deletion in the gene SLC9A6 was identified as the causative agent in an X-linked mental retardation family displaying seizures, ataxia and other symptoms resembling those of Angelman syndrome [8]. Subsequently, three other mutations in the gene were identified in patients with genetically unexplained Angelman syndrome-like phenotypes, including a large pedigree designated as having Christianson syndrome [43]. SLC9A6 encodes NHE6, a member of the Na+/H+ exchanger (NHE) family that mediates the electroneutral exchange of H+ for Na+ or K+ across membranes. NHE6 localizes to early/recycling endosomes, and has been hypothesized to regulate endosomal pH by exporting H+ out of the endosome lumen [9]. The endocytic pathway is characterized by a luminal acidified pH gradient, ranging from the neutral pH of newly internalized vesicles to pH 5.0 and lower in lysosomes, and this pH gradient is essential for many transport processes. The yeast homologue of NHE6, Nhx1, regulates endosomal pH and trafficking, and a direct link between the pH regulation and the trafficking function has been demonstrated [10], [11]. Apart from the endosomal localization, NHE6 has been found to transiently localize to the plasma membrane [9], mediated by an interaction with Rack1 [12].

The identified 6-bp mutation in SLC9A6 results in deletion of amino acids 255–256 of the NHE6 protein. These two residues are situated in a highly conserved region of the predicted transmembrane domain 7 of NHE6, and Glutamate 255 is essential for the ion transport in the NHE family members NHE1 and NHE8 [13], [14], [15]. In order to understand the molecular basis for the protein dysfunction caused by the 2-aa deletion, we have characterized the cellular behaviour of the deletion protein. Our results indicate that NHE6Δ255–256 is unstable in the cell and is rapidly degraded by two independent mechanisms; proteasomal degradation and lysosomal degradation. Moreover, we present evidence supporting the hypothesis that NHE6 and the closely related NHE9 are involved in regulation of endosomal pH.

Section snippets

Plasmid constructs and siRNA

The NHE6 construct (NHE6.0 isoform b) containing a GFP and a HA tag was a kind gift from Dr. R. Rao (The John Hopkins University School of Medicine, Baltimore, USA). The NHE6Δ255–256-GFP was generated from NHE6-GFP by quickchange mutagenesis PCR introducing the 6 bp deletion (nucleotide 764–769 in NHE6.0 ORF) and a silent restriction enzyme site for construct verification using the primer sequence: 5′-GCACTTCTTTTTGGGGTCCTCAATGATGCTG-3′. A pcDNA3.1 hemagglutinin epitope (HA) tagged K44A mutant

NHE6Δ255–256-GFP is unstable and rapidly degraded, partly dependent on the proteasomal degradation machinery

Deletion of amino acids 255 and 256 of the NHE6 protein causes X-linked mental retardation with several neurological features similar to those in Angelman syndrome. In order to characterize the mechanism behind the protein dysfunction, we generated a NHE6Δ255–256 construct on the basis of the NHE6.0 splice form containing a C-terminal GFP tag for visualization. First, the stability of the protein was assessed by transiently expressing NHE6wt (wild type)-GFP or NHE6Δ255–256-GFP in HeLa cells for

Discussion

Mutations in the SLC9A6-gene, encoding the ion exchanger NHE6, have recently been shown to cause an X-linked mental retardation syndrome with phenotypical characteristics similar to those of Angelman syndrome [8]. The initial mutation identified in this work was a deletion of 6 bp resulting in the lack of amino acids 255 and 256 of the NHE6 protein product (Supplementary Fig. 1). In the present study we have characterized this deletion protein, and observed that it is highly unstable due to

Acknowledgments

C.R. is a postdoctoral fellow of the Norwegian Cancer Society and is supported by Lars Henry Lie and Dagmar and Reidar Jensen's Legacy for Cancer Research. This work was also supported by the Research Council of Norway. We thank Dr. R. Rao and Dr. S. L. Schmid for kindly providing plasmids and Dr. T. Johansen for antibodies.

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