Sodium-potassium ATPase 1 subunit is a molecular partner of Wolframin, an endoplasmic reticulum protein involved in ER stress

Abstract

Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein, Wolframin. Although its precise functions are unknown, Wolframin deficiency increases ER stress, impairs cell cycle progression and affects calcium homeostasis. To gain further insight into its function and identify molecular partners, we used the WFS1-C-terminal domain as bait in a yeast two-hybrid screen with a human brain cDNA library. Na+/K+ ATPase beta1 subunit was identified as an interacting clone. We mapped the interaction to the WFS1 C-terminal and transmembrane domains, but not the N-terminal domain. Our mapping data suggest that the interaction most likely occurs in the ER. We confirmed the interaction by co-immunoprecipitation in mammalian cells and with endogenous proteins in JEG3 placental cells, neuroblastoma SKNAS and pancreatic MIN6 beta cells. Na+/K+ ATPase beta1 subunit expression was reduced in plasma membrane fractions of human WFS1 mutant fibroblasts and WFS1 knockdown MIN6 pancreatic beta-cells compared with wild-type cells; Na+/K+ ATPase alpha1 subunit expression was also reduced in WFS-depleted MIN6 beta cells. Induction of ER stress in wild-type cells only partly accounted for the reduced Na+/K+ ATPase beta1 subunit expression observed. We conclude that the interaction may be important for Na+/K+ ATPase beta1 subunit maturation; loss of this interaction may contribute to the pathology seen in Wolfram syndrome via reductions in sodium pump alpha1 and beta1 subunit expression in pancreatic beta-cells.

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome

“…S5). It has been shown that WFS1 interacts with the Na + /K + ATPase β1 subunit and the expression of WFS1 parallels that of Na + /K + ATPase β1 subunit in a variety of settings, suggesting that WFS1 may function as an ion channel or regulator of existing channels (42). Further studies on this topic would be necessary to completely understand the etiology of Wolfram syndrome.…”

“…It has been shown that WFS1-deficient pancreatic β cells have high baseline ER stress levels and impaired insulin synthesis and secretion. Thus, WFS1-deficient β cells are susceptible to ER stress mediated cell death (5,6,32,(40)(41)(42). The functions of WFS2 are still not clear.…”

Neurexin-1α Contributes to Insulin-containing Secretory Granule Docking

“…Plasma membrane was separated from cytosolic fractions as previously described by differential centrifugation and detergent/aqueous partitioning using the Plasma Membrane Protein Extraction kit (BioVision) per the manufacturer's instructions (38). Proteins were quantified using the D C Protein Assay (Bio-Rad).…”

Identification of homozygous WFS1 mutations (p.Asp211Asn, p.Gln486*) causing severe Wolfram syndrome and first report of male fertility

“…[8][9][10][11] Other roles, such as regulation of ER stress and protein biosynthesis, modification/ folding, have also been suggested for Wolframin. [12][13][14][15] Wolframin is an 890 amino acid oligomeric protein with a molecular mass of 100 kDa. 8,9 Although many mutations have been identified in the WFS1 gene, most affected patients carry a mutation, either insertion, deletion, nonsense or missense, in exon 8, the largest exon of WFS1.…”