title: Dual functional significance of calcineurin homologous protein 1 binding to Na(+)/H(+) exchanger isoform 1.
abstract: Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na(+)/H(+) exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246-C254, 2007). However, Pang et al. (J Biol Chem 276: 17367-17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsushita
authors.forename: Hiroo
authors.initials: H
authors.lastname: Tanaka
authors.forename: Keiji
authors.initials: K
authors.lastname: Mitsui
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kanazawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006031:Glycosylation; D000882:Haplorhini; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D021381:Protein Transport; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D054875:Ubiquitination
chemical_list: D002135:Calcium-Binding Proteins; D017923:Sodium-Hydrogen Exchangers