Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components.
Activation of the superoxide-generating NADPH oxidase system of human neutrophils involves the assembly of several neutrophil components, some located on the plasma membrane and others in the cytosol. It has recently been established that one of the required components for NADPH oxidase activity is the GTP-binding protein Rac. To further investigate the role of Rac in the NADPH oxidase system, studies were carried out to determine its subcellular distribution in resting and activated human neutrophils. In resting cells, Rac and an associated guanine nucleotide regulatory factor, GDP dissociation inhibitor (GDI), were located only in the cytosol, along with other known oxidase factors, p47-phox and p67-phox. After activation of neutrophils with phorbol 12-myristate 13-acetate or formyl-methionyl-leucyl-phenylalanine, Rac was translocated from the cytosol to the plasma membrane, and this translocation corresponded temporally with the translocation of p47-phox and p67-phox and with the generation of superoxide. GDI remained localized to the cytosol, suggesting activation of the oxidase involved dissociation of the Rac-GDI complex prior to Rac translocation. Determination of the quantities of cytosolic factors associated with the plasma membrane indicated that Rac, p47-phox, and p67-phox are translocated to the plasma membrane simultaneously in equimolar amounts, but that the membrane-associated cytochrome b was present at 3-4-fold molar excess. These findings suggest that Rac may play a role in assembly of the active NADPH oxidase complex.
Quinn
M T
MT
Department of Microbiology, Montana State University, Bozeman 59717.
Evans
T
T
Loetterle
L R
LR
Jesaitis
A J
AJ
Bokoch
G M
GM
D001692:Biological Transport; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate