Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558.
When the neutrophil NADPH oxidase is activated to generate superoxide, the cytosolic components, p47phox, p67phox, and the GTP-binding protein Rac, become stably associated with the plasma membrane. The translocation of p47phox and p67phox is dependent on the presence in the membrane of the flavocytochrome b558, itself composed of two subunits, gp91phox and p22phox. In this study we have quantitated Rac1 and Rac2 in human neutrophils and show that > 96% of the Rac protein is Rac2 and that chronic granulomatous disease (CGD) neutrophils contain normal levels of the protein. We used a cell-free translocation system as well as intact normal and CGD neutrophils to determine whether the translocation of Rac2 is dependent upon the presence of the other oxidase components. When cell-free reactions contained any combination of normal, p47phox-deficient or p67phox-deficient cytosol and normal or flavocytochrome b558-deficient membranes, the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-dependent association of Rac2 with the re-isolated membranes was not significantly different from the control mixture of normal membranes and cytosol. In intact CGD neutrophils lacking p47phox or p67phox and stimulated with phorbol myristate acetate the translocation of Rac2 was also normal, and we conclude that Rac translocation can occur independently of the cytosolic NADPH oxidase components. In contrast, in the absence of flavocytochrome b558 from intact X-chromosome linked CGD neutrophils, Rac2 translocation was reduced to only 25% of the control value. On the basis of these observations we propose that while Rac2 can bind to a site distinct from either gp91phox or p22phox, it depends upon an interaction with the flavocytochrome b558 for maximal stability in the membrane.
Heyworth
P G
PG
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Bohl
B P
BP
Bokoch
G M
GM
Curnutte
J T
JT
D001692:Biological Transport; D003573:Cytochrome b Group / Q000097:blood; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006105:Granulomatous Disease, Chronic / Q000097:blood; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D009252:NADPH Dehydrogenase / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000097:blood*; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins