Macrophage NADPH Oxidase Flavocytochrome b Localizes to the Plasma Membrane and Rab11-Positive Recycling Endosomes

• Published in: The Journal of immunology (1950) Vol. 182; no. 4; pp. 2325 - 2339
• Main Authors: Casbon, Amy-Jo, Allen, Lee-Ann H, Dunn, Kenneth W, Dinauer, Mary C
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15.02.2009
• Subjects: Animals; Blotting, Western; Cell Membrane - enzymology; Cell Membrane - immunology; CHO Cells; Cricetinae; Cricetulus; Cytochrome b Group - immunology; Cytochrome b Group - metabolism; Endosomes - immunology; Endosomes - metabolism; Flow Cytometry; Fluorescent Antibody Technique; Humans; Image Processing, Computer-Assisted; Macrophages - enzymology; Macrophages - immunology; Mice; Microscopy, Confocal; NADPH Oxidases - genetics; NADPH Oxidases - immunology; NADPH Oxidases - metabolism; Protein Transport - immunology; rab GTP-Binding Proteins - immunology; rab GTP-Binding Proteins - metabolism; Transgenes
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.0803476

Flavocytochrome b(558), 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 gp91(phox) (NOX2) and a smaller subunit, p22(phox). 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 gp91(phox) and p22(phox), 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 p22(phox) and gp91(phox) 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.