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* Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Department of Microbiology and Immunology and
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202;
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, IN 46202; and
Inflammation Program, Departments of Medicine and Microbiology, University of Iowa and the Veterans Affairs Medical Center, Coralville, IA 52241
Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91phox and p22phox, we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22phox and gp91phox subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health (NIH) Grants R01 HL45635 (to M.C.D.), TK32 DKI07519 (to A.J.C.), R01 AI073835 (to L.A.A.), and R01 DK51098 (to K.W.D.); a Veterans Affairs Merit Review Grant (to L.A.A.); NIH Grant P30 CA082709 to the Indiana University Simon Cancer Center, which provides partial support for the Indiana University Flow Cytometry and Indiana Center for Biological Microscopy Cores; and funding from the Riley Children’s Foundation (to M.C.D.).
2 Address correspondence and reprint requests to Dr. Mary C. Dinauer, Indiana University School of Medicine, Herman B. Wells Center, 1044 West Walnut Street, Room 402A, Indianapolis, IN 46202. E-mail address: mdinauer{at}iupui.edu
3 Abbreviations used in this paper: ER, endoplasmic reticulum; BM, bone marrow; BMDM, bone marrow-derived macrophage; CFP, cyan fluorescent protein; CHO, Chinese hamster ovary; SOZ, serum-opsonized zymosan; YFP, yellow fluorescent protein.
4 The online version of this article contains supplemental material.
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