Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.
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.
Casbon
Amy-Jo
AJ
Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Department of Microbiology and Immunology, Indianapolis, IN 46202, USA.
Allen
Lee-Ann H
LA
Dunn
Kenneth W
KW
Dinauer
Mary C
MC
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D003573:Cytochrome b Group / Q000276:immunology / Q000378:metabolism*; D011992:Endosomes / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008264:Macrophages / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D021381:Protein Transport / Q000276:immunology; D019076:Transgenes; D020691:rab GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D003573:Cytochrome b Group; D019255:NADPH Oxidases; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C036888:cytochrome b558