Translocation of p21rac2 from Cytosol to Plasma Membrane Is Neither Necessary nor Sufficient for Neutrophil NADPH Oxidase Activity

Abstract

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21roc2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTPγS stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTPγS-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically. Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21roc2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTPγS stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTPγS-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically. Neutrophils and other phagocytic cells have the capacity to generate superoxide anion (O2⨪) and related toxic oxygen species that are critical to host defense against microorganisms and play a role in autoimmune tissue damage. The multimeric enzyme responsible for O2⨪ production, NADPH oxidase, is latent in resting neutrophils. Upon stimulation with a variety of inflammatory agonists, an active oxidase complex is transiently assembled at the plasma/phagolysosome membrane (1Morel F. Doussiere J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (526) Google Scholar). Studies of patients with the X-linked form of chronic granulomatous disease characterized by a complete absence of neutrophil NADPH oxidase permitted the identification of the first component of the oxidase, cytochrome b558 (2Segal A.W. Jones O.T.G. Webster D. Allison A.C. Lancet. 1978; ii: 446-449Abstract Scopus (119) Google Scholar), a heterodimeric glycoprotein intrinsic to neutrophil plasma membrane (PM). 1The abbreviations used are: PM, plasma membrane; CS, cytosol; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GDPβS, guanyl-5′-yl thio-phosphate; GMP-PNP, guanyl-5′-yl imidodiphosphate; ATPγS, adenosine 5′-O-(3-thiotriphosphate); PMA, phorbol 12-myristate 13-acetate; GDI, GDP dissociation inhibitors; AFC, N-acetyl-S-trares,trans-farnesyl-l-cysteine; AGGC, N-acetyl-S-all trares-geranylgeranyl-l-cysteine. Over the last decade, the development of cell-free systems for studying NADPH oxidase activation (3Bromberg Y. Pick E. Cell Immunol. 1984; 88: 213-221Crossref PubMed Scopus (249) Google Scholar, 4Curnutte J.T. J. Clin. Invest. 1985; 75: 1740-1743Crossref PubMed Scopus (199) Google Scholar, 5McPhail L.C. Shirley P.S. Clayton C.C. Snyderman R. J. Clin. Invest. 1985; 75: 1735-1739Crossref PubMed Scopus (228) Google Scholar, 6Bromberg Y. Pick E. J. Biol. Chem. 1985; 260: 13539-13545Abstract Full Text PDF PubMed Google Scholar) has permitted the identification of other proteins associated with the oxidase (7Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (569) Google Scholar). The minimum complement of proteins required to assemble an active NADPH oxidase in a fully recombinant system include cytochrome b658 and three cytosolic factors designated p47phox, p67phox, and p21rac2 (8Rotrosen D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar). Activation of the latent NADPH oxidase appears to involve translocation of the cytosolic factors to the PM. Both p47phox and p67phox contain src homology 3 (SH3) domains (9Lomax K.J. Leto T.L. Nunoi H. Gallin J.I. Malech H.L. Science. 1989; 245: 409-412Crossref PubMed Scopus (235) Google Scholar, 10Volpp B.D. Nauseef W.M. Donelson J.E. Moser D.R. Clark R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7195-7199Crossref PubMed Scopus (229) Google Scholar, 11Leto T.L. Lomax K.J. Volpp B.D. Nunoi H. Sechler J.M. Nauseef W.M. Clark R.A. Gallin J.I. Malech H.L. Science. 1990; 248: 727-730Crossref PubMed Scopus (298) Google Scholar) known to mediate protein-protein interactions in other signaling systems. In resting neutrophils, p47phox has been found exclusively in the cytosol (CS) whereas p67phox has been localized to both the CS and a Triton X-100-insoluble fraction thought to consist of cytoskeleton (12Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (328) Google Scholar, 13Nauseef W.M. Volpp B.D. McCormick S. Leidal K.G. Clark R.A. J. Biol. Chem. 1991; 266: 5911-5917Abstract Full Text PDF PubMed Google Scholar, 14Woodman R.C. Ruedi J.M. Jesaitis A.J. Okamura N. Quinn M.T. Smith R.M. Curnutte J.T. Babior B.M. J. Clin. Invest. 1991; 87: 1345-1351Crossref PubMed Scopus (123) Google Scholar). A stable complex of p47phox and p67phox in the CS of resting neutrophils has been reported (15Park J.W. Ma M. Ruedi J.M. Smith R.M. Babior B.M. J. Biol. Chem. 1992; 267: 17327-17332Abstract Full Text PDF PubMed Google Scholar, 16Iyer S.S. Pearson D.W. Nauseef W.M. Clark R.A. J. Biol. Chem. 1994; 269: 22405-22411Abstract Full Text PDF PubMed Google Scholar). Stimulation of neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) promotes phosphorylation of p47phox (17Heyworth P.G. Segal A.W. Biochem. J. 1986; 239: 723-731Crossref PubMed Scopus (71) Google Scholar, 18Reibman J. Korchak H.M. Vosshall L.B. Haines K.A. Rich A.M. Weissmann G. J. Biol. Chem. 1988; 263: 6322-6328Abstract Full Text PDF PubMed Google Scholar, 19Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Abstract Full Text PDF PubMed Google Scholar) and induces its redistribution to the PM and Triton X-100-insoluble fractions (12Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (328) Google Scholar, 13Nauseef W.M. Volpp B.D. McCormick S. Leidal K.G. Clark R.A. J. Biol. Chem. 1991; 266: 5911-5917Abstract Full Text PDF PubMed Google Scholar, 20El Benna J. Ruedi J.M. Babior B.M. J. Biol. Chem. 1994; 269: 6729-6734Abstract Full Text PDF PubMed Google Scholar). The same stimuli promote association of p67phox with the PM (12Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (328) Google Scholar, 13Nauseef W.M. Volpp B.D. McCormick S. Leidal K.G. Clark R.A. J. Biol. Chem. 1991; 266: 5911-5917Abstract Full Text PDF PubMed Google Scholar). Studies using neutrophils from patients with chronic granulomatous disease lacking either cytochrome b558, p47phox, or p67phox suggest that cytochrome b558 is required for translocation of both p47phox and p67phox and that p47phox can translocate to the PM in the absence of p67phox p67phox translocation requires p47phox P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. H. Clark R.A. J. Clin. Invest. 1991; 87: PubMed Scopus Google Scholar). p47phox and p67phox have been to translocate to the PM in cell-free systems in response to and GTPγS N. 1992; PubMed Scopus Google Scholar, J. Babior B.M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, A. A. Segal A.W. Biochem. J. 1994; PubMed Scopus Google Scholar), required for NADPH oxidase p21rac2 is a of the of that regulate a variety of by a and a active McCormick F. 1991; PubMed Scopus Google Scholar). p21rac2 is by and that it to membranes S. Biochem. 1992; PubMed Scopus Google Scholar). of the and of proteins can by association with proteins known GDP dissociation Moreover, the of of this of proteins membranes and appears to the M. A. N. Y. 1991; Google Scholar, Biol. 1993; PubMed Scopus Google Scholar). The cytosolic proteins of the NADPH oxidase, from A. Pick E. A. N. Segal A.W. 1992; Scopus Google Scholar) and p21rac2 from neutrophils P.G. Curnutte J.T. Science. 1991; PubMed Scopus Google Scholar) were both found to with p21rac2 an for the that NADPH oxidase at the neutrophil PM by translocation from the We reported that in a cell-free system, GTPγS stimulated membrane translocation of neutrophil not in which GTPγS stimulated of p21rac2 R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar). a cell-free system and neutrophil we have the that translocation of p21rac2 from CS to PM NADPH oxidase activity by of the oxidase We found that p21rac2 translocation is of stimulated and is for NADPH oxidase of these have been Weissmann G. Clin. 1993; Scholar, Weissmann G. Clin. 1994; Scholar). were from from from from U. S. A and were from from and from and were and R.A. 1990; Scopus Google Scholar). GDPβS, GTPγS, GMP-PNP, and were from to and were from A that from to by and a F. J.M. F. H. J. Biochem. 1993; PubMed Scopus Google Scholar) by to recombinant p47phox and p67phox by Malech were from and were from from Neutrophils were from of by by and of A. J. Clin. Invest. Google Scholar). Neutrophils were in Neutrophil fractions PM, and were by by we have Haines K.A. Weissmann G. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). membranes were J.T. Biochem. PubMed Scopus Google Scholar). Neutrophil CS against of to in to were and in of fractions were in at and CS at to in translocation were from neutrophils by and at by D. J. Cell Biol. PubMed Scopus Google Scholar). were and in with and used The of of cytoplasts with an of O2⨪ Neutrophil fractions were by and in 3 with in The were with in for and for and and for at and with A or for at were a used an with A. The and the proteins were by and by The cell-free translocation with and without of PM In the of CS with of membranes and to to with or without GTPγS, arachidonate, or other for a of or the with of and the and fractions were by for in a using a The were to by and by and the that by in A PM reisolated of CS with of PM in a of at for the with an of and the a in and at for at in an CS the the and PM reisolated from the of the CS to by and by of the reisolated PM. by of the and the of the cytosolic oxidase in the or reisolated PM of the reisolated PM in and to of of with and of which superoxide The NADPH oxidase activity of the reisolated PM the of the in at in a using the and of membrane by the Neutrophils were with PMA, or to CS and PM for p47phox, and p67phox by and were in of for to of or were to of and and with three from a were and for of PM in the cell-free of the CS for p47phox, and p67phox by of the PM The of cytosolic oxidase component in the CS and PM by and the associated with the PM for of the PM without of CS, GTPγS, or used to cell-free NADPH oxidase activity for reisolated neutrophil PM. of neutrophil fractions by and that of p21rac2 of PM and of CS the of the = were at for and by of p21rac2 in the In the presence of GTPγS, the of p21rac2 found in the to Both the and GTPγS-stimulated association of p21rac2 with the the PM In contrast, a of both cytosolic p47phox and p67phox in the in the absence of PM GTPγS the of p47phox and whereas this cell-free system not for membrane association of p47phox and p67phox, it a GTPγS-stimulated in the association of neutrophil cytosolic p21rac2 with PM and reported membrane translocation of p21rac2. of GMP-PNP, stimulated p21rac2 although to a extent GTPγS at the same GTPγS in p21rac2 translocation. the of did not the extent of translocation. activity GTPγS-stimulated membrane translocation of p21rac2 by and stable for at membrane association of p21rac2 at in the absence of GTPγS and NADPH oxidase activation in cells and in cell-free systems is of cell-free membrane translocation with a of p21rac2 membrane in a in to membrane translocation of GTPγS stimulated of the same R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar), we stimulated is required for membrane translocation. the and the the GTPγS-stimulated membrane translocation of p21rac2 did p21rac2 translocation. activation of p21rac2 with GTPγS can promote its membrane association without of of and in a GTPγS-stimulated membrane translocation to neutrophil the of three other proteins and the were in the absence or presence of GTPγS other of the of and were cytosolic in neutrophils. GTPγS stimulated membrane translocation of both and to an extent to that for p21rac2. found exclusively in the CS in the absence or presence of In contrast, a in the that has been reported to associated with cytochrome of the neutrophil oxidase M.T. Jesaitis A.J. 1989; PubMed Scopus Google Scholar), found in both PM and membranes not CS Haines K.A. Weissmann G. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). GTPγS did not the of in the cell-free membrane translocation is a by several of the of proteins in neutrophils. We GTPγS-stimulated p21rac2 translocation to The membranes of an of intrinsic PM proteins cytochrome b558 N. J.M. Clark R.A. J. Cell Biol. PubMed Scopus Google Scholar) an inflammatory membranes are to the PM the of PM, membranes GTPγS-stimulated membrane translocation of p21rac2 In contrast, although of neutrophil cytosolic p21rac2 associated with membranes at in this association in the presence of neutrophil plasma and membranes contain a that GTPγS-stimulated membrane translocation of p21rac2. GTPγS-stimulated p21rac2 translocation of CS PM without it is that a is NADPH oxidase activity of reisolated PM with membrane translocation of p47phox and not p21rac2 or CS and PM were in the absence or presence of and the of GTPγS the at the with of and the PM reisolated a The reisolated PM for NADPH oxidase activity the superoxide of without of NADPH The reisolated PM and CS were for p47phox p67phox p21rac2 and by by The extent of membrane translocation is the of in the PM are of and or and NADPH oxidase activity and cytosolic membrane translocation in the presence of of 0.97, and for p47phox, p67phox, and the the membrane translocation of cytosolic oxidase and NADPH oxidase activity in a cell-free system we translocation to the PM of p47phox, and p67phox and compared it to the capacity of the same membranes to generate O2⨪ In to neutrophil PM and CS, and an are required for cell-free NADPH oxidase activity (3Bromberg Y. Pick E. Cell Immunol. 1984; 88: 213-221Crossref PubMed Scopus (249) Google Scholar, 4Curnutte J.T. J. Clin. Invest. 1985; 75: 1740-1743Crossref PubMed Scopus (199) Google Scholar, 5McPhail L.C. Shirley P.S. Clayton C.C. Snyderman R. J. Clin. Invest. 1985; 75: 1735-1739Crossref PubMed Scopus (228) Google Scholar, 6Bromberg Y. Pick E. J. Biol. Chem. 1985; 260: 13539-13545Abstract Full Text PDF PubMed Google Scholar, P.G. Curnutte J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). PM and CS were in the presence of of GTPγS and in the absence or presence of the of found to promote O2⨪ in cell-free of p47phox and p67phox from we reisolated the PM a a translocation of neutrophil CS and PM in the presence of GTPγS with or without for at The reisolated PM used for both of p47phox, and p67phox and for of NADPH oxidase The oxidase reisolated PM by the of NADPH the cell-free without of CS, arachidonate, or that a the extent of cytosolic component PM translocation and the capacity of the PM to O2⨪ of GTPγS, reisolated PM to generate O2⨪ from the translocation The NADPH oxidase activity of PM to CS and to the of GTPγS the translocation NADPH oxidase activity to a at GTPγS to at GTPγS = data suggest in cell-free system, GTPγS and promote of a stable NADPH oxidase in the PM, GTPγS is required for O2⨪ from reisolated PM active is with the that PM from neutrophils stimulated with an active oxidase in the absence of or M. Curnutte J.T. Babior B.M. J. Clin. Invest. PubMed Scopus Google Scholar). In the absence of arachidonate, p47phox p67phox translocated to the reisolated PM at of GTPγS A and In the presence of arachidonate, the of p47phox and p67phox in the reisolated PM from to and to at GTPγS = for both p47phox and to at GTPγS A and the of both p47phox and p67phox in reisolated PM paralleled the NADPH oxidase activity of the = 0.94 and = for p47phox and p67phox, In to p47phox and p67phox, not required for GTPγS-stimulated translocation of p21rac2. a p21rac2 translocation in the presence of at of GTPγS, the were not Moreover, the response of p21rac2 translocation to GTPγS did not a to to membrane-associated at GTPγS-stimulated p21rac2 translocation did not correlate with NADPH oxidase = The for p21rac2 translocation that for either O2⨪ or p47phox and p67phox translocation. GTPγS, a that of O2⨪ and p47phox and p67phox p21rac2 translocation not in cell-free system, NADPH oxidase activation from membrane translocation of p21rac2 of p21rac2 to the PM not required for GTPγS-stimulated oxidase stable membrane translocation of p21rac2 not for NADPH oxidase activity neutrophil translocated to reisolated PM in a Moreover, at GTPγS, the in translocation stimulated by other neutrophil cytosolic and translocated to reisolated PM in a only in the presence of the of the membrane translocation of cytosolic oxidase to NADPH oxidase activation in an system that stimulated with inflammatory agonists, we neutrophils and we a translocation of from CS to PM in neutrophils stimulated with and not were Neutrophil cytoplasts several neutrophils. in response to the same neutrophils and are to regulate NADPH oxidase in the same cytoplasts consist of PM of PM and CS and by in a were stimulated with or PMA, and were for at PM and CS were with The of p47phox, and p67phox in the PM compared to the capacity of the same membranes to O2⨪ of NADPH without of CS, GTPγS, or In response to the NADPH oxidase activity in PM to a of of cytochrome and to of cytochrome by The of both p47phox and p67phox in the PM of cytoplasts transiently from to and to with to of NADPH oxidase activity and In contrast, the of p21rac2 in the PM only from to in response to stimulated NADPH oxidase activity in PM although to a the activity stimulated by of cytochrome activity, NADPH oxidase activity in PM not and sustained for at stimulated PM translocation of both p47phox and p67phox from to and to with to for NADPH oxidase activation and In contrast, did not stimulate p21rac2 translocation to the PM of cytoplasts in the GTPγS-stimulated cell-free system, whereas the extent and of p47phox and p67phox membrane translocation paralleled NADPH oxidase activity in and membrane translocation of p21rac2 did Moreover, stimulated stable NADPH oxidase activity in membranes without translocation of p21rac2. We have of GTPγS-stimulated translocation of p21rac2 to membranes in a cell-free system R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar) by that this is stable and its and membranes from neutrophils not GTPγS-stimulated p21rac2 to of translocated to the PM in the absence of or a related required for cell-free NADPH oxidase this that p21rac2 translocation to the PM is to promote oxidase We have that GTPγS membrane translocation of other of the of proteins including and Moreover, the PM translocation of of these these only and p21rac2 have activity in the neutrophil cell-free NADPH oxidase A. Pick E. A. N. Segal A.W. 1992; Scopus Google Scholar, P.G. Curnutte J.T. Science. 1991; PubMed Scopus Google Scholar, S. M. Nunoi H. Y. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, P.G. Curnutte J.T. Biol. 1993; PubMed Scopus Google Scholar, Malech H.L. D. Leto T.L. 1993; PubMed Scopus Google Scholar). the capacity for and translocation to the PM not to p21rac2 is with in neutrophil cytosol A. Pick E. A. N. Segal A.W. 1992; Scopus Google Scholar, P.G. Curnutte J.T. Science. 1991; PubMed Scopus Google Scholar) of R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar), data suggest that promotes dissociation of p21rac2 from and association with PM. of for in this GDP dissociation is the in were to to dissociation of the GDP activity of A. N. J. Y. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar) have to The presence of a in neutrophil PM active for this is to for dissociation both the and of the related and have in for A. J. 1993; PubMed Scopus Google Scholar). in which by neutrophil GTPγS did not the of p21rac2 and in cytosol to that is to an membrane is both and have been to for membrane of the not the of M. A. N. Y. 1991; Google Scholar) and proteins Biol. 1993; PubMed Scopus Google Scholar, S. A. Y. M. Y. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar), that the of proteins that and membranes membranes and that neutrophil plasma and membranes p21rac2 membranes did that translocation requires a an to neutrophil we to a cell-free system of neutrophil PM and CS with the for the of p21rac2 we GTPγS-stimulated of p21rac2 R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar). p21rac2 is the same in which it to PM, that the are that that activity in neutrophils not a of of NADPH oxidase R. Weissmann G. Science. 1993; PubMed Scopus Google Scholar). several we have that GTPγS-stimulated of p21rac2 is not required for membrane translocation. The is to neutrophil is associated with the PM Weissmann G. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In cell-free system, the and PM translocation of p47phox and p67phox correlated with the capacity of the reisolated PM to O2⨪ (R = 0.94 and = 0.97, respectively). In contrast, translocation of p21rac2 and NADPH oxidase activity did not correlate (R = against a role for in which the NADPH oxidase activity of cell-free system of p21rac2 cell-free does not a role for membrane-associated p21rac2 in oxidase data suggest that cell-free NADPH oxidase activity does not with the extent of p21rac2 translocation. data suggest that GTPγS has an p47phox and p67phox membrane translocation of the component of the NADPH oxidase, p21rac2. are with of J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) translocation of p47phox and p67phox in a cell-free system lacking cytosolic p21rac2. have reported p21rac2 translocation in stimulated neutrophils. Quinn M.T. Jesaitis A.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) reported that p47phox, and p67phox translocate to the PM in in response to both and with that paralleled O2⨪ whereas the of O2⨪ and several the oxidase factors to in the PM Abo A. A. Segal A.W. Biochem. J. 1994; PubMed Scopus Google Scholar) reported that stimulated the translocation of only and of p67phox, and p47phox, in cells whereas stimulated the translocation of of 3 with a dissociation of p21rac2 and p67phox not p47phox, without of NADPH oxidase Benna Benna J. Ruedi J.M. Babior B.M. J. Biol. Chem. 1994; 269: 6729-6734Abstract Full Text PDF PubMed Google Scholar) reported that stimulated translocation of and of neutrophil p47phox, and p67phox, to the PM the of p47phox, and p67phox in the CS of resting neutrophils are P.G. Curnutte J.T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), of the translocation. H. G. Biochem. 1994; PubMed Scopus Google Scholar) to membrane translocation of p21rac2 in response to either or We found of cytosolic oxidase translocation in neutrophils to of the required to fractions by and to the stimulation to oxidase to Neutrophil cytoplasts several a system with which to translocation to the PM. with to of neutrophils stimulation with or D. J. Cell Biol. PubMed Scopus Google Scholar, H.M. D. J.P. Rich A.M. Weissmann G. Proc. Natl. Acad. Sci. S. A. PubMed Scopus Google Scholar) and not only of the required to assemble an active oxidase the required signaling and neutrophils with and cytoplasts and CS and PM. Whereas PM from neutrophils has been reported to contain active NADPH oxidase not stimulation M. Curnutte J.T. Babior B.M. J. Clin. Invest. PubMed Scopus Google Scholar), we found that PM from cytoplasts NADPH oxidase activity, the of the Moreover, the NADPH oxidase activity transiently in with the and in the of in both neutrophils and cytoplasts stimulated with data the first of a NADPH oxidase in a only did we a in both p47phox and p67phox associated with the translocation transient and to of NADPH oxidase activity for the first a for the oxidase and its In contrast, although the p21rac2 of PM with the of the translocation to relatively the of p21rac2 to p47phox and p67phox in neutrophil CS, data that for of p21rac2 translocated to the membrane in response to a stimulation with and of and that although stimulated both NADPH oxidase activity and translocation of p47phox and p67phox in cytoplasts from by p21rac2 translocation not of p21rac2 associated with the PM, the of translocation to this membrane that of p21rac2 to the PM is not required for NADPH oxidase p21rac2 has been to a required for NADPH oxidase activity in cells A. J. G. G. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and cell-free systems A. Pick E. A. N. Segal A.W. 1992; Scopus Google Scholar, P.G. Curnutte J.T. Science. 1991; PubMed Scopus Google Scholar, S. M. Nunoi H. Y. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, P.G. Curnutte J.T. Biol. 1993; PubMed Scopus Google Scholar, Malech H.L. D. Leto T.L. 1993; PubMed Scopus Google Scholar, P.G. J. Curnutte J.T. Biol. 1993; PubMed Scopus Google Scholar). of proteins is required for membrane S. Biochem. 1992; PubMed Scopus Google Scholar), the of P.G. Curnutte J.T. Biol. 1993; PubMed Scopus Google Scholar) that is not required for the activity of the cell-free NADPH oxidase that the membrane capacity of p21rac2 is of its activity the data this by the membrane translocation of p21rac2 from NADPH oxidase activation in both a cell-free system and in neutrophil The for p21rac2 in a fully recombinant cell-free oxidase system of (8Rotrosen D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar) that or of the other oxidase p67phox not p47phox has been to with the of p21rac2 in a D. Abo A. Segal A.W. A. Science. 1994; 265: PubMed Scopus Google Scholar). A of oxidase that cytosolic oxidase in and an component of the active oxidase (7Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (569) Google Scholar, M.T. Jesaitis A.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). data are with this and suggest that p21rac2 cytochrome b558, p47phox, or activity without the active oxidase We D. H. A. and J. for and recombinant We are to for data the of p47phox, and p67phox in neutrophil We Weissmann for critical of