The endoplasmic reticulum chaperone calnexin is a NADPH oxidase NOX4 interacting protein

  • Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

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Author:Kim-Kristin Prior, Ilka WittigORCiD, Matthias LeisegangORCiDGND, Jody Groenendy, Norbert WeißmannGND, Marek MichalakORCiD, Pidder Jansen-DürrORCiDGND, Ajay M. ShahORCiD, Ralf BrandesORCiDGND
Pubmed Id:
Parent Title (English):The journal of biological chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Place of publication:Bethesda, Md.
Document Type:Article
Date of Publication (online):2016/11/28
Date of first Publication:2016/02/09
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2016/11/28
Page Number:16
First Page:7045
Last Page:7059
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license.
Institutes:Medizin / Medizin
Exzellenzcluster / Exzellenzcluster Makromolekulare Komplexe
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung 4.0