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Biochem. J. (2007) 403 (97–108) (Printed in Great Britain)

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Critical roles for p22^phox in the structural maturation and subcellular targeting of Nox3

Yoko NAKANO*†, Botond BANFI*‡, Algirdas J. JESAITIS§, Mary C. DINAUER, Lee-Ann H. ALLEN*† and William M. NAUSEEF*†¹

*Inflammation Program, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52241, U.S.A., †Department of Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52241, U.S.A., ‡Department of Anatomy and Cell Biology, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52241, U.S.A., §Department of Microbiology, Montana State University, Bozeman, MT 59717, U.S.A., and Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ~50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22^phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22^phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22^phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22^phox. Moreover, the glycosylation and maturation of Nox3 required p22^phox expression, suggesting that p22^phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22^phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

Key words: flavocytochrome b₅₅₈, glycosylation, haem spectrum, NADPH oxidase 3 (Nox3), Nox organizer 1 (NOXO1), p22^phox.

Abbreviations used: CHO, Chinese-hamster ovary; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFP, enhanced GFP; HEK-293 cells, human embryonic kidney cells; Nox, NADPH oxidase; NoxA1, Nox activator 1; NoxO1, Nox organizer 1; NP40, Nonidet P40; PNGase F, peptide:N-glycosidase F; PX domain, Phox homology domain; RNAi, RNA interference; SA, succinylacetone (4,6-dioxoheptanoic acid); SH3, Src homology 3; siRNA, small interfering RNA; SOD, superoxide dismutase; TNT, transcription and translation; WGA, wheat germ agglutinin.

¹To whom correspondence should be addressed, at Inflammation Program, Department of Medicine, University of Iowa, D160 MTF, 2501 Crosspark Road, Coralville, IA 52241, U.S.A. (email william-nauseef@uiowa.edu).

Received 2 June 2006/30 November 2006; accepted 1 December 2006

Published as BJ Immediate Publication 1 December 2006, doi:10.1042/BJ20060819

The Biochemical Society, London ©2007