title: Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability.
abstract: Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that also localizes to secretory granules in pancreatic beta cells. Although its precise functions are unknown, WFS1 protein deficiency affects the unfolded protein response, intracellular ion homeostasis, cell cycle progression and granular acidification. In this study, immunofluorescent and electron-microscopy analyses confirmed that WFS1 also localizes to secretory granules in human neuroblastoma cells. We demonstrated a novel interaction between WFS1 and the V1A subunit of the H(+) V-ATPase (proton pump) by co-immunoprecipitation in human embryonic kidney (HEK) 293 cells and with endogenous proteins in human neuroblastoma cells. We mapped the interaction to the WFS1-N terminal, but not the C-terminal domain. V1A subunit expression was reduced in WFS1 stably and transiently depleted human neuroblastoma cells and depleted NT2 (human neuron-committed teratocarcinoma) cells. This reduced expression was not restored by adenoviral overexpression of BiP (immunoglobulin-binding protein) to correct the ER stress. Protein stability assays demonstrated that the V1A subunit was degraded more rapidly in WFS1 depleted neuroblastoma cells compared with wild-type; however, proteosomal inhibition did not restore the expression of the V1A subunit. Cell cycle assays measuring p21(cip) showed reduced levels in WFS1 depleted cells, and an inverse association between p21(cip) expression and apoptosis. We conclude that WFS1 has a specific interaction with the V1A subunit of H(+) ATPase; this interaction may be important both for pump assembly in the ER and for granular acidification.
authors.affiliation: Section of Medical and Molecular Genetics, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Seley
authors.initials: S
authors.lastname: Gharanei
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Zatyka
authors.forename: Dewi
authors.initials: D
authors.lastname: Astuti
authors.forename: Janine
authors.initials: J
authors.lastname: Fenton
authors.forename: Attila
authors.initials: A
authors.lastname: Sik
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Nagy
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Barrett
mesh_terms: D017209:Apoptosis / Q000235:genetics; D002352:Carrier Proteins; D002453:Cell Cycle / Q000235:genetics; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D017494:Proton Pumps / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D017494:Proton Pumps; C114987:wolframin protein; C581697:ATP6V1A protein, human; D025262:Vacuolar Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase