Targeting of Rac1 to the Phagocyte Membrane Is Sufficient for the Induction of NADPH Oxidase Assembly*

The superoxide (O 2 .)-generating NADPH oxidase com- plex of phagocytes consists of a membrane-associated fla-vocytochrome (cytochrome b 559 ) and four cytosolic pro- teins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O 2 . production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b 559 . This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47 phox , p67 phox , and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O 2 . production, in a cell-free system containing phagocyte membrane vesicles and p67 phox , in the absence of an activating amphiphile and of p47 phox . Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same condi-tions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b 559 , incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, en-zyme-linked into the Bam HI and Eco RI sites of the baculovirus transfer vector pBacPAK-His1 (CLONTECH). Rac1 cDNA was introduced into the baculovirus genome using the BaculoGold system (PharMingen). Positive colonies from plaque assays were used for the production of virus stocks that served for the infection of Sf9 cell suspension cultures in serum-free medium. Infected Sf9 cells, grown for 72 h in an orbital shaker at 27 °C, were sedimented and disrupted by sonication in buffer A (20 M MgCl 2 in the presence of protease inhibitors (Complete; Molec- ular Biochemicals), and membranes and cytosol by centrifugation Themembranes three resuspended the same and at Thawed solubilized in buffer A, supplemented 21 m M sodium cholate (Sigma), and the His 6 -tagged prenylated Rac1 was batch-purified on Talon metal affinity resin beads (CLONTECH), using 50 m M imidazole, in buffer A containing 21 m M sodium cholate, for elution. Prenylation of Rac1 was confirmed by its ability to form a heterodimer with Rho GDI, this being identified by gel filtration on Superdex 75 and anion exchange chromatography on Mono Q. His 6 -tagged nonprenylated Rac1 purified from the cytosol by the same technique, except the use of 50 m M imidazole in buffer A, lacking cholate.

The production of reactive oxygen species represents the major microbicidal mechanism of professional phagocytes. The primordial oxygen radical is superoxide (O 2 . ), 1 and it is pro-duced by the NADPH-derived one-electron reduction of molecular oxygen, by an enzyme complex known as the NADPH oxidase (referred to here as "oxidase"; reviewed in Refs. 1 and 2). This consists of a membranal heterodimeric flavocytochrome (cytochrome b 559 ), comprising two subunits, gp91 phox and p22 phox , and four cytosolic proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac (Rac1 or -2). Elicitation of O 2 . production in vivo involves the stimulus-dependent translocation of some or all cytosolic components to the plasma membrane and their assembly with cytochrome b 559 . Oxidase assembly can be induced in vitro by exposing a mixture of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac to a critical concentration of an anionic amphiphile (3,4). Rac1 or -2 is absolutely required for oxidase assembly in the amphiphile-activated cell-free system (5,6). It is also clearly involved in O 2 . production in intact phagocytes, as shown by the inhibitory effect of Rac antisense oligonucleotides (7) and by selective defects in O 2 . production by neutrophils of Rac2-deficient mice (8) and of a patient with an inhibitory mutation in Rac2 (9). It is the consensus opinion that, in the course of oxidase assembly, Rac is translocated to the membrane (10), although lack of translocation (11) or the lack of relevance of translocation to assembly (12) was also claimed. In the cytosol, Rac is found as a C-terminally prenylated (geranylgeranylated) protein (13), forming a heterodimer with the regulatory protein GDP dissociation inhibitor for Rho (Rho GDI) (5). Dissociation of prenylated Rac from Rho GDI was proposed to be an obligatory step preceding translocation of Rac from the cytosol to the membrane (14 -16). The role of Rac in oxidase assembly was studied extensively in the semirecombinant amphiphile-activated cell-free system (4). Both nonprenylated (5,17) and prenylated (17)(18)(19) Rac are capable of supporting oxidase activation in vitro. It was recently suggested (20) that, whereas membrane association of prenylated Rac (Rac1 or -2) is mediated principally by hydrophobic interaction between the geranylgeranyl tail and membrane lipids, nonprenylated Rac1 binds by electrostatic interaction between a C-terminal polybasic region (21) and negative charges on the membrane. We intended to test the hypothesis that binding of Rac to the membrane is a crucial event in the initiation of oxidase assembly. We found indeed that prenylated, but not nonprenylated, Rac1 initiates oxidase assembly and NADPH-dependent O 2 .
production in a cell-free system, containing phagocyte membranes and p67 phox , in the absence of an amphiphilic activator and of p47 phox . This demonstrates that, under certain conditions, targeting of Rac1 to the phagocyte membrane is sufficient for the initiation of oxidase assembly and suggests that the principal (or only) role of Rac is to act as a carrier for p67 phox , from the cytosol to the membrane.

EXPERIMENTAL PROCEDURES
Preparation of Phagocyte Membranes and Membrane Vesicles-Phagocyte membranes were prepared from guinea pig peritoneal macrophages elicited by the injection of mineral oil, as described (3). The membranes were solubilized by 40 mM n-octyl-␤-D-glucopyranoside (octyl glucoside), and membrane vesicles were prepared by dialysis against buffer lacking detergent, as described (22). These vesicles were found to have a M r exceeding 40 ϫ 10 6 by gel filtration on a Superose 6 fast protein liquid chromatography (FPLC) column.
Purification and Relipidation of Cytochrome b 559 -Cytochrome b 559 was purified partially from octyl glucoside-solubilized guinea pig macrophage membranes by affinity chromatography on a HiTrap Heparin column (5 ml; Amersham Pharmacia Biotech), essentially as described before (23) with the following modifications: (a) batch absorption by DEAE-Sepharose was replaced by passage through a HiTrap DEAE-Sepharose column (5 ml; Amersham Pharmacia Biotech) placed in line ahead of the heparin column, and (b) the hydroxylapatite chromatography step was omitted. The purified cytochrome b 559 was relipidated with soybean phosphatidylcholine (PC) (10 -20% phosphatidylcholine, type II-S, product 5638; Sigma), at a concentration of 200 g/ml, as described before (23), and vesicles were prepared by dialysis of relipidated cytochrome b 559 against detergent-free buffer for 18 h at 4°C. The partially purified cytochrome b 559 preparations had a specific content of 4 -6 nmol of heme/mg of protein.
Preparation of PC Vesicles-Soybean PC (the same type as the one used for relipidation of cytochrome b 559 ) was dissolved, at a concentration of 4 mg/ml, in the buffer also used for solubilization of macrophage membranes (22), containing 40 mM octyl glucoside. Vesicles were generated by the removal of detergent by dialysis, following the procedure described for the preparation of vesicles from solubilized phagocyte membranes (22).
Preparation of Recombinant Oxidase Components-p67 phox and p47 phox were prepared in Sf9 cells, infected with baculovirus carrying cDNAs of the oxidase components (human), as described (24). Recombinant nonprenylated Rac1 (human) and Rho GDI (bovine) were isolated from Escherichia coli as fusion proteins with glutathione S-transferase (GST), as described (24). In some experiments, we used bacterially expressed p67 phox (human); this was also isolated from E. coli as a fusion protein with GST. cDNA of truncated p67 phox (residues 1-212) was obtained by polymerase chain reaction, and the fragment was cloned into the pGEX-2T vector (Amersham Pharmacia Biotech). The GST-p67 phox -(1-212) fusion protein was expressed in E. coli (DH5␣competent cells; Life Technologies) and purified by affinity chromatography on glutathione-agarose (Sigma).
Construction and Isolation of His 6 -tagged Prenylated and Nonprenylated Rac1-The cDNA for Rac1 was extracted from the pGEX-2T clone, used for the expression of the protein in E. coli, by digestion with the restriction enzymes BamHI and EcoRI. In order to express His 6 -Rac1 in insect cells, the cDNA was cloned into the BamHI and EcoRI sites of the baculovirus transfer vector pBacPAK-His1 (CLONTECH). Rac1 cDNA was introduced into the baculovirus genome using the BaculoGold system (PharMingen). Positive colonies from plaque assays were used for the production of virus stocks that served for the infection of Sf9 cell suspension cultures in serum-free medium. Infected Sf9 cells, grown for 72 h in an orbital shaker at 27°C, were sedimented and disrupted by sonication in buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl 2 ) in the presence of protease inhibitors (Complete; Roche Molecular Biochemicals), and membranes and cytosol were separated by centrifugation at 43,000 ϫ g for 1 h at 4°C. The membranes were washed three times in buffer A, resuspended in the same buffer, and frozen at Ϫ75°C. Thawed membranes were solubilized in buffer A, supplemented with 21 mM sodium cholate (Sigma), and the His 6 -tagged prenylated Rac1 was batch-purified on Talon metal affinity resin beads (CLONTECH), using 50 mM imidazole, in buffer A containing 21 mM sodium cholate, for elution. Prenylation of Rac1 was confirmed by its ability to form a heterodimer with Rho GDI, this being identified by gel filtration on Superdex 75 and anion exchange chromatography on Mono Q. His 6 -tagged nonprenylated Rac1 was purified from the cytosol by the same technique, except for the use of 50 mM imidazole in buffer A, lacking sodium cholate.
Cdc42Hs-His 6 -tagged prenylated Cdc42Hs was a kind gift from Dr. R. A. Cerione (Cornell University). It was produced by baculoviral expression in Sf21 cells and purified from the membrane fraction. The protein was found to react on immunoblot with a polyclonal anti-Cdc42Hs antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; product sc-87). Its functional integrity was ascertained by its ability to form a complex with Rho GDI. Nucleotide Exchange-Prenylated and nonprenylated Rac1 (in buffers containing 5 mM MgCl 2 ) were subjected to nucleotide exchange with GTP␥S or GDP␤S, at a free Mg 2ϩ concentration of 0.5 M, attained by adding 9 mM EDTA, at a 30-fold molar excess of nucleotide over protein, by incubation for 30 min at 30°C. The exchange was stabilized by adding MgCl 2 to a final concentration of 30 mM. Unbound nucleotide was removed by applying the protein to a HiTrap desalting column (5 ml; Amersham Pharmacia Biotech), eluting this with buffer A, supplemented or not with 21 mM sodium cholate, and collecting the proteinrich fractions. In cases in which Rac preparations were intended for use in gel filtration experiments, the HiTrap column elution buffer was supplemented with 20% (v/v) glycerol, and sodium cholate was replaced by 3.12 mM octyl glucoside (modified from Ref. 20). In both 21 mM sodium cholate and 3.12 mM octyl glucoside plus 20% glycerol, prenylated Rac was maintained in soluble form.
Assembly of Oxidase in a Cell-free System in the Absence of Amphiphile-Assembly of the oxidase complex was assessed by measuring NADPH-dependent O 2 . production in a cell-free system, consisting of total membrane or purified cytochrome b 559 vesicles (5 nM cytochrome b 559 heme), recombinant prenylated Rac1 (0 -500 nM; subjected to nucleotide exchange to either GTP␥S or GDP␤S), and recombinant p67 phox (0 -1600 nM), in 96-well microplates, in a volume of 200 l of assay buffer (25) per well. No free GTP␥S was added to the reaction. In some experiments, p47 phox was included, at a concentration of 300 nM. The mixtures were incubated for 90 s at 25°C, in the absence of an amphiphilic activator, and O 2 . generation was initiated by the addition of NADPH (240 M). O 2 . production was measured by the kinetics of cytochrome c reduction, as described before (25). Specificity of cytochrome c reduction was controlled by its inhibition by superoxide dismutase (Sigma). Amphiphile-dependent cell-free oxidase activation was performed using the same concentrations of components as indicated above but with the addition of p47 phox (300 nM) and lithium dodecyl sulfate (LiDS) (130 M).
In Line Fluorescence Assay-Prenylated or nonprenylated Rac1 were subjected to nucleotide exchange with the fluorescent analogues 2Ј-(or 3Ј)-O-(N-methylanthraniloyl) guanosine 5Ј-triphosphate (mant-GTP) or mant-GDP (26), as described for nonfluorescent nucleotides, except for not removing unbound nucleotides. Rac1 labeled with fluorescent analogue (1500 pmol) was mixed or not with either membrane vesicles (250 pmol of cytochrome b 559 heme) or PC vesicles (0.6 mg of lipid), injected in a Superose 12 HR 10/30 FPLC gel filtration column (Amersham Pharmacia Biotech), and eluted with a buffer consisting of 65 mM sodium, potassium phosphate, pH 7.0, 2 mM NaN 3 , 1 mM EGTA, 1 mM MgCl 2 , and 1 M FAD. Its composition is, in essence, identical to reaction buffer used in the cell-free oxidase assay (25). Chromatography was performed on an HPLC system (Waters), at a flow rate of 0.2 ml/min, at 4°C, and the fluorescent signal (excitation ϭ 356 nm; emission ϭ 445 nm) was measured continuously, by passing the column eluate through a spectrofluorometer (model FP-770; Jasco), fitted with an HPLC flow cell.
Assembly of the Oxidase Complex Studied by Gel Filtration-Vesicles prepared from solubilized membrane (250 pmol of cytochrome b 559 heme) were mixed with prenylated or nonprenylated Rac1 (1500 pmol), with p67 phox (1500 pmol), or with both Rac1 and p67 phox (1500 pmol of each), in a total volume of 0.6 ml of column elution buffer, in the absence of an activating amphiphile. After incubation for 10 min at 4°C, the mixture was injected in a Superose 12 gel filtration column, and chromatography was performed as described above, except for the fact that the absorbance of the eluate was measured continuously at 280 and 413 nm, by an HP 1040A diode array detector (Hewlett-Packard). Throughout chromatography, 0.6-ml fractions were collected. The elution volume of membrane vesicles was identified by a major peak in the 413-nm absorbance profile, representing a measure of the amount of cytochrome b 559 . The amounts of membrane-bound Rac1 and p67 phox were determined by an NADPH oxidase activity reconstitution assay (see below), and the relative amounts of membrane-bound and free p67 phox were measured by an enzyme-linked immunosorbent assay (ELISA).
NADPH Oxidase Activity Reconstitution Assay-In this assay, 2 the membrane association of a certain cytosolic oxidase component was 2 Y. Gorzalczany, N. Sigal, and E. Pick, manuscript in preparation. demonstrated by the ability of a sample, taken from fractions containing the membrane vesicles, to produce O 2 . when supplemented with the two other cytosolic oxidase components and activated by LiDS. First, the amount of cytochrome b 559 present in membrane vesicles was determined by addition to a mixture of p47 phox , p67 phox , and nonprenylated Rac1-GTP␥S. Membrane-associated Rac1 was quantified by the addition to a mixture of p47 phox and p67 phox , and membrane-associated p67 phox was quantified by addition to a mixture of p47 phox and nonprenylated Rac1-GTP␥S. In all assays, 20 l of column fraction were added to 180 l of reaction mixture, containing the supplementing cytosolic components, each at 100 nM, and 130 M LiDS. Incubation was for 90 s, and O 2 . production was initiated by the addition of 240 M NADPH.
ELISA-With the purpose of confirming results obtained by the assay described above, free and membrane-bound p67 phox were measured by ELISA (27). Column fractions were diluted 1:10 in elution buffer and added in volumes of 100 l, in triplicate, to 96-well microplates (MaxiSorp C96, Nunc). The proteins were allowed to adhere to the wells for 18 h at 4°C, and the plates were processed as described in Ref. 27. p67 phox was detected by a goat polyclonal antiserum to recombinant human p67 phox , diluted 1:1000, followed by peroxidase-conjugated rabbit anti-goat IgG (H ϩ L) (code 305-035-003; Jackson Immu-noResearch), diluted 1:10,000. Peroxidase was quantified by the 3,3Ј,5,5Ј-tetramethylbenzidine liquid substrate system (product T8665; Sigma), followed by 0.5 M H 2 SO 4 , and absorbance at 450 nm was read in a Spectramax 340 microplate reader (Molecular Devices).
Inhibition of Oxidase Assembly-Three categories of compounds were studied for an inhibitory effect on amphiphile-independent oxidase assembly, involving prenylated Rac1: (a) Rho GDI; (b) PC vesicles, and (c) a synthetic peptide, corresponding to residues 177-191 of Rac1 (Ͼ70% pure; Chiron Technologies). Potential inhibitors were added to the assay mixture either from the beginning, being present during the 90 s of incubation (assembly) preceding the addition of NADPH, or after incubation of the oxidase components for 5 min, to be followed, after another 90 s, by the addition of NADPH. Controls were always run in parallel, by adding a volume of buffer equal to that of the inhibitory compound tested. In all experiments, the concentration of membrane corresponded to 5 nM cytochrome b 559 heme; Rac1 was in the GTP␥Sbound form (unless specified otherwise), and both Rac1 and p67 phox were at a concentration of 300 nM. The design was identical for comparative experiments, involving prenylated or nonprenylated Rac1, p67 phox , p47 phox , and activation by LiDS (130 M).

Amphiphile-independent Activation of NADPH Oxidase by Prenylated Rac1
The initial aim of this investigation was to reexamine the influence of the state of prenylation of Rac1 on its ability to support cell-free assembly of the oxidase complex. Earlier work seemed to indicate that prenylation of Rac is essential for oxidase activation (18), a result that disagreed with reports describing activation by nonprenylated Rac, exchanged to GTP␥S (5,17). A more recent report clearly indicates that prenylated Rac is a superior activator because of its higher affinity for the phagocyte membrane, mediated by hydrophobic interaction between the geranylgeranyl group and membrane lipids (20). Common to all of these findings is the requirement for an anionic amphiphile and for the presence of both p47 phox and p67 phox , for activation to take place.
We found that, surprisingly, a cell-free system consisting of phagocyte membrane vesicles and recombinant prenylated Rac1, p67 phox , and p47 phox produces O 2 . upon the addition of NADPH, in the absence of any activator. Next, we found that the presence of p47 phox is not required, its absence leading to only a minor reduction in the amount of O 2 . produced; however, no O 2 . is produced if p67 phox is replaced by p47 phox , either in the absence or presence of amphiphile. The level of oxidase activation by prenylated Rac1 in the absence of amphiphile and p47 phox , as expressed by O 2 . production, is comparable with that customary in the complete amphiphile-activated cell-free system. Amphiphile and p47 phox -independent oxidase activation, of the same or superior intensity, is also occurring when membrane vesicles are replaced by partially purified cytochrome b 559 incorporated in PC vesicles. The characteristics of this novel oxidase activation system are summarized in Table I. Thus, the absence or heat inactivation (95°C for 30 min) of any of the three obligatory components of the reaction results in a total lack of O 2 . production. Prenylation of Rac1 is a sine qua non condition for activity; nonprenylated Rac1 is incapable of inducing O 2 . production, whether derived from E. coli (Table I;   with the concentration of prenylated Rac1 (Fig. 1, A and B) and p67 phox (Fig. 1C). It is of interest that, in the presence of either total membrane or purified cytochrome b 559 and p67 phox , both the GTP␥S-and the GDP␤S-bound forms of prenylated Rac1 are active, with Rac1-GTP␥S being only marginally more effective (Fig. 1, A and B). The possible reasons for this finding are addressed under "Discussion." Recombinant p67 phox proteins, isolated from baculovirus-infected Sf9 cells or from E. coli are both capable of cooperation with prenylated Rac, and p67 phox truncated at residue 212 is as potent as full-length protein (Fig.  1C), in accordance with the finding that the Rac-binding domain is located within the first 199 residues of p67 phox (28). No GTPase other than Rac1 or -2 was found capable of supporting NADPH oxidase activation in the canonical amphiphile-dependent system (17). To find out whether this also applied to amphiphile-independent oxidase activation, we tested prenylated (geranylgeranylated) Cdc42Hs, a member of the Rho subfamily of GTPases closely related to Rac. The cellfree system consisted of vesicles derived from solubilized membrane (5 nM cytochrome b 559 heme), prenylated Cdc42Hs, exchanged to GTP␥S (0 -100 nM), and p67 phox (300 nM

Prenylated but Not Nonprenylated Rac1 Binds to Phagocyte Membrane Vesicles
These findings are compatible with a model, first proposed by Lambeth (20), in which the extent of membrane association of Rac is a key factor in determining the level of oxidase activation. To test this model, we designed a system permitting the direct measurement of the binding of prenylated and nonprenylated Rac1 to membrane vesicles. In this technique, prenylated and nonprenylated Rac1 were labeled with the fluorescent GTP analogue, mant-GTP, or with mant-GDP; mixed with membrane vesicles; and subjected to gel filtration on a Superose 12 column. Free Rac1 and Rac1 associated with membrane vesicles were identified by their elution volumes, using an in line fluorescence detector. Free prenylated Rac1 elutes at 14 ml, corresponding to a M r of 21,000 ( Fig. 2A); the fluorescence peak starting at 25 ml represents mant-GTP not bound to Rac1. When prenylated Rac1 is mixed with membrane vesicles and injected in the column, the bulk of Rac1 elutes with the membrane in the exclusion volume (7.6 ml, corresponding to molecules with a M r Ͼ 2 ϫ 10 6 ) (Fig. 2B). Recruitment of prenylated Rac1 to the membrane was independent of the nature of bound nucleotide; Rac1-mant-GTP and Rac1-mant-GDP attach to membrane in equal amounts (results not shown). Nonprenylated Rac1 does not bind to membrane vesicles, most of it eluting as free Rac1 (Fig. 2C). Fig. 2D illustrates an experiment in which artificial protein-free PC vesicles (replacing phagocyte membrane vesicles) were mixed with prenylated Rac1 and subjected to gel filtration. This approach is based on the work of Araki et al. (29), showing that prenylated RhoA binds to phospholipid-coated beads by hydrophobic interaction. It is apparent that the majority of prenylated Rac1 elutes in association with PC vesicles, indicating that binding of prenylated Rac1 to membrane vesicles is mediated by Rac1lipid interaction. Preincubation of prenylated Rac1 with Rho GDI before mixing with either phagocyte membrane (Fig. 2E) or PC (Fig. 2F) vesicles, followed by gel filtration, results in a reduction in the relative amounts of prenylated Rac1 associated with membrane or PC vesicles and the appearance of a new fluorescence peak, eluting at 12.4 ml, corresponding to the M r of the Rac1-Rho GDI heterodimer (52,000). 3 A comparison of E and F in Fig. 2 indicates that Rho GDI is less able to compete for Rac1 with PC vesicles than with membrane vesicles, suggesting that prenylated Rac1 has a higher affinity for artificial soybean PC vesicles than for native phagocyte membrane vesicles (the total amounts of lipid present in the two types of vesicles injected in the column were comparable). Variations in relative fluorescence readings between individual experiments are probably due to variable losses of prenylated Rac1 during 3 The size of the Rac1-Rho GDI heterodimer is larger than expected because of the presence of a remaining fusion linker in recombinant Rho GDI, prepared by thrombin digestion of the Rho GDi-GST fusion protein. experimental steps preceding injection in the column (see the higher recovery of nonprenylated Rac1; Fig. 2C). Therefore, we based our conclusions on comparing the relative sizes of fluorescence peaks within individual experiments rather than between experiments.

Membrane Association of Prenylated Rac1 Leads to Amphiphile-independent Translocation of p67 phox to the Membrane
The simplest explanation for the induction of oxidase assembly by prenylated Rac1, in the absence of amphiphile and p47 phox , is that prenylated Rac1 is responsible for the translocation of p67 phox to the membrane by virtue of the double affinity of Rac1 for p67 phox and for membrane lipid. To demonstrate this in quantitative terms, membrane vesicles were mixed with prenylated or nonprenylated Rac1 and p67 phox and injected in a Superose 12 column. The collected fractions were analyzed for the presence of membrane-associated p67 phox by the NADPH oxidase activity reconstitution assay and by ELISA (Fig. 3). These assays demonstrate that (a) prenylated, but not nonprenylated, Rac1 binds to membrane vesicles containing cytochrome b 559 (Fig. 3, A, C, and D), confirming the results illustrated in Fig. 2; (b) in the absence of Rac1 or in the presence of nonprenylated Rac1, only a minor fraction of p67 phox translocates to membrane vesicles (Fig. 3, B, D, and E), and (c) in the presence of prenylated Rac1, a significantly larger amount of p67 phox becomes associated with membrane vesicles (Fig. 3, C and E). The quantitative difference between p67 phox recruited to the membrane, in the absence and presence of prenylated Rac1, appears to be more pronounced when assessed by the NADPH oxidase activity reconstitution assay (Fig. 3, comparison of B with C) than by ELISA (Fig. 3E). A possible explanation for this is that, whereas the activity assay offers a direct measure of components in solution, the ELISA results are influenced by secondary parameters, such as the affinity of components for the surface of the plate wells, the geometry of their attachment, and the accessibility of antigenic epitopes to the detecting antibody.
The mere addition of NADPH to samples taken from fractions containing membrane vesicles with bound Rac1 and p67 phox (such as shown in Fig. 3C) results in spontaneous O 2 .
production, demonstrating that these fractions contain a fully assembled and activated oxidase complex (Fig. 4A). Adding NADPH to fractions containing membrane vesicles preincubated with p67 phox only (such as shown in Fig. 3B) or with nonprenylated Rac1 and p67 phox (such as shown in Fig. 3D) does not elicit O 2 . production (Fig. 4A).
Incomplete oxidase assembly, however, can be brought to completion; thus, the addition of p67 phox and NADPH to fractions containing membrane vesicles with bound Rac1 (such as shown in Fig. 3A) results in O 2 . production; the addition of p47 phox has no effect (Fig. 4B). Interestingly, the ability to bring FIG. 2. Prenylated, but not nonprenylated, Rac1 binds to membrane and PC vesicles, in the absence of amphiphile; Rho GDI competes with membrane and PC vesicles for binding of prenylated Rac1. Prenylated or nonprenylated Rac1 were subjected to nucleotide exchange to mant-GTP, mixed with membrane or PC vesicles, and separated by FPLC gel filtration on a Superose 12 column. In some experiments, prenylated Rac1 was preincubated with Rho GDI for 10 min at 30°C at a 3-fold molar excess of Rho GDI over Rac1, before mixing with membrane or PC vesicles. The eluates were monitored in line for the fluorescent signal of mant-GTP. Bound Rac1 represents the fraction bound to membrane or PC vesicles, eluting in the exclusion volume. Free Rac1 represents the fraction not bound to vesicles, detected at the expected elution volume (M r of 21,000). Rac1-Rho GDI represents the heterodimer of Rac1 and Rho GDI, detected at an elution volume corresponding to a M r of 52,000. The excess of mant-GTP left from nucleotide exchange is retained on the column, due to hydrophobic interaction. A, prenylated Rac1 injected in the absence of membrane vesicles. B, prenylated Rac1 mixed with membrane vesicles. C, nonprenylated Rac1 mixed with membrane vesicles. D, prenylated Rac1 mixed with PC vesicles. E, prenylated Rac1 preincubated with Rho GDI before the addition to membrane vesicles. F, prenylated Rac1 preincubated with Rho GDI before the addition to PC vesicles. The panels illustrate representative individual experiments out of three experiments performed for each combination of components.
incomplete oxidase assembly to completion is dependent on the order of association of the cytosolic components with the membrane. Thus, in the presence of sodium cholate, at concentrations above the critical micellar concentration (8 mM), p67 phox is incorporated in membrane vesicles and can be detected in such vesicles, upon gel filtration on Superose 12, in a fully functional state by the NADPH oxidase activity reconstitution assay ( Fig.  4C; filled squares). However, the addition of prenylated Rac1 and NADPH to fractions containing membrane-associated p67 phox does not result in O 2 . production (Fig. 4C, filled triangles), in contrast to the situation, illustrated in Fig. 4B, in which the addition of p67 phox and NADPH to membrane vesicles with bound Rac1 led to amphiphile-independent O 2 . generation. That this difference is not due to eventual damage to the membrane or p67 phox by sodium cholate is shown by the ability of the same membrane vesicles with bound p67 phox to be active in the NADPH oxidase activity reconstitution assay. Furthermore, in experiments identical to those illustrated in Fig. 4B, but in which prenylated Rac1-GTP␥S is mixed with membrane vesicles in the presence of 9 mM sodium cholate before gel filtration on Superose 12, the outcome is the same; the addition of p67 phox and NADPH results in O 2 . production (results not shown).

Amphiphile-independent NADPH Oxidase Activation Involving Prenylated Rac1 Is Subject to Inhibition by Agents Competing with the Membrane for Binding of Rac1
Inhibition by Rho GDI-Rac1 was originally identified as a component of the oxidase complex by its purification from macrophage cytosol as a heterodimer with Rho GDI (5,14). Both native and recombinant Rac1-Rho GDI dimers are potent activators of O 2 . production in the amphiphile-dependent cellfree system (14,30). We found that, in amphiphile-independent oxidase activation by prenylated Rac1, Rho GDI acts in a diametrically opposite fashion, as an inhibitor (Fig. 5A). Inhibition by Rho GDI is evident whether Rac1 is in the GTP-or GDPbound form (results not shown). Upon supplementation of the system with p47 phox and LiDS, Rho GDI is no longer inhibitory. Inhibition by Rho GDI is maximal when it is added simultaneously with the components of the cell-free system; adding Rho GDI 5 min after the initiation of oxidase assembly leads to a marked reduction in its inhibitory effect (Fig. 5C). Interference by Rho GDI with oxidase activation was found with both total membrane and purified cytochrome b 559 preparations (results not shown). The ability of Rho GDI to prevent amphiphileindependent oxidase activation by prenylated Rac1 is in good agreement with the experiments, illustrated in Fig. 2, E and F, showing that Rho GDI is capable of counteracting the recruitment of Rac1 to both membrane and PC vesicles.
Inhibition by PC Vesicles-The fact that prenylated Rac1 binds to PC vesicles (Fig. 2D) suggested that such vesicles might compete with bona fide membrane vesicles for Rac1. Indeed, the addition of PC vesicles to a mixture of membrane vesicles, prenylated Rac1, and p67 phox , in the absence of amphiphile, causes a marked, dose-dependent inhibition of oxidaseactivation (Fig.5B).PCvesicleshavenoeffectonamphiphiledependent activation involving prenylated or nonprenylated Rac1, p67 phox , and p47 phox . A calculation of the relative amounts of added PC to the amount of membrane phospholipid present in the reactions (based on data appearing in Ref. 22) shows that, at 40 g/ml PC (which results in almost complete inhibition of activation), the ratio is close to 1:1. PC vesicles, like Rho GDI, are inhibitory only when added together with the components of the cell-free system; PC added 5 min after the initiation of assembly exerts a much lesser inhibitory effect (Fig. 5D). Inhibition of oxidase activation by PC vesicles also takes place when membrane is replaced by purified cytochrome b 559 (results not shown).
Effect of Polybasic Rac1 Peptide-Membrane association of Rac1 also involves electrostatic interaction between a polybasic region at the C terminus of Rac1 and negative charges on the membrane (20,21), as shown by the ability of synthetic peptides, incorporating the polybasic region, to interfere with amphiphile-dependent oxidase activation (21,31). We investigated the effect of a 15-mer peptide, corresponding to residues 177-191 of Rac1 (LCPPPVKKRKRKCLL) and containing six basic residues, on amphiphile-independent oxidase activation supported by prenylated Rac1. This (21) and related (32) polybasic Rac1 peptides were found to be potent inhibitors of the amphiphile-activated cell-free system, containing nonprenylated Rac1 (IC 50 , in the 1-2 M range). Rac1 peptide 177-191 exerts no inhibitory effect on amphiphile-independent oxidase assembly involving prenylated Rac1 and p67 phox , up to a concentration of 40 M peptide. Thus, percentage inhibition of oxidase activation by 40 M Rac1 peptide 177-191 is Ϫ5.5 Ϯ 3.9 (mean Ϯ S.E. of three experiments; negative arithmetic value is the result of minor enhancement of activation). DISCUSSION We describe the induction of oxidase assembly and consequent NADPH-driven O 2 . production in a cell-free system, consisting of phagocyte membranes, recombinant p67 phox , and recombinant prenylated Rac1, in the absence of an amphiphilic activator and of p47 phox . Identical results are obtained when total membrane is replaced by partially purified and relipidated cytochrome b 559 , suggesting that the only functional protein contributed by the membrane is cytochrome b 559 and that "nonphysiological" lipids can successfully replace native phagocyte lipids. Prenylated Cdc42Hs, a GTPase exhibiting about 70% overall identity with Rac1 and -2, is incapable of supporting amphiphile-independent oxidase activation. Prenylated, but not nonprenylated, Rac1 was found to attach avidly to phagocyte membrane vesicles and to artificial PC vesicles. Binding of prenylated Rac1 to membrane vesicles could be demonstrated by the use of Rac labeled with a fluorescent marker and by an NADPH oxidase activity reconstitution assay. Attachment occurs in the absence of amphiphile, is independent of the type of nucleotide (GTP or GDP) bound to Rac, and appears to be mediated exclusively by the geranylgeranyl tail.
In mixtures of membrane vesicles, prenylated Rac1 and p67 phox , binding of prenylated Rac1 to membrane is accompanied by the parallel binding of a fraction of p67 phox . Only minor binding of p67 phox to membrane is seen in the absence of prenylated Rac1 or in the presence of nonprenylated Rac1. Cotranslocation of prenylated Rac1 and p67 phox to the membrane results in the formation of an assembled oxidase complex, which produces O 2 . upon the addition of NADPH, in the absence of free cytosolic components and amphiphile. The sequence of events in this form of oxidase assembly cannot yet be established with certainty. However, it is likely that translocation of prenylated Rac1 to the membrane precedes that of p67 phox . Support for this possibility is provided by the ability of exogenous p67 phox to join membrane vesicles that bound prenylated Rac1 to generate a fully assembled O 2 . -generating complex, as opposed to the inability of achieving the same result when binding of p67 phox to the membrane precedes that of Rac1. A seemingly unusual feature of the system described in this report is that the GDP-bound form of prenylated Rac1 is only , and 300 nM p47 phox (filled squares). C, membrane vesicles were mixed with p67 phox in the presence of 9 mM sodium cholate and separated by gel filtration on a Superose 12 column. First, membrane-associated p67 phox (filled squares) was quantified by the NADPH oxidase activity reconstitution assay, as described under "Experimental Procedures." Next, amphiphile-independent oxidase activity was assayed by the addition to 20-l samples from the same fractions of 300 nM prenylated Rac1-GTP␥S, in the absence of amphiphile (filled triangles). All panels illustrate individual experiments out of three to four experiments performed for each combination of components.
slightly less active than the GTP-bound form in supporting oxidase activation, whether in the presence of membrane or purified cytochrome b 559 . In fact, this is quite similar to our earlier finding that the GDP-bound form of prenylated Rac1, in complex with Rho GDI ( 1 ), is a potent activator of oxidase in the amphiphile-activated cell-free system (33). We also showed, recently, that the relative abilities of the GTP-and GDP-bound forms of nonprenylated Rac1 to support amphiphile-dependent oxidase activation are related to the concentrations of p47 phox and p67 phox present; at a 200 nM concentration of these components, the difference in V max between Rac1-GTP␥S and Rac1-GDP␤S was marginal (25). Indeed, it might be of significance that, in the present work, the activities of prenylated Rac1-GTP␥S and Rac1-GDP␤S were compared at a concentration of 300 nM p67 phox . Oxidase activation by prenylated Rac1, in the absence of amphiphile and p47 phox , is prevented by agents, such as Rho GDI and PC vesicles, that compete with the membrane for binding of Rac1 via its geranylgeranyl tail. These results and the lack of inhibition by a polybasic Rac1 peptide point to the key importance of hydrophobic bonds in the binding of Rac1 to the membrane. Both Rho GDI and PC are inhibitory only when added before the completion of oxidase assembly. The fact that they are ineffectual after assembly indicates either that, once membrane-bound, Rac cannot be dislodged or that, on the contrary, the continuous presence of Rac in the complex is not required for O 2 . generation to proceed.
A remarkable characteristic of oxidase activation by prenylated Rac1 is the lack of requirement for activating amphiphile and p47 phox , contrasting with the absolute dependence of the canonical cell-free system on both components. The preponderant view is that amphiphiles cause a conformational change in p47 phox , leading to the conversion of intramolecular bonds in p47 phox to intermolecular bonds between p47 phox and the p22 phox subunit of cytochrome b 559 (34). This is expected to lead to the secondary translocation to the membrane of p67 phox , in complex with p47 phox . Amphiphile-independent oxidase activation was described in the past but required the presence of both p47 phox and p67 phox and was conditional on either C-terminal truncation of both components (35) or on phosphorylation of p47 phox by protein kinase C (36). On the other hand, p47 phox was shown not to be required for oxidase activation in vitro, provided that p67 phox and Rac were present at high (Ͼ1 M) concentrations (37,38), and a recent report mentions that, under these conditions, activation also occurred in the absence of amphiphile (39). All this points to prenylated Rac1 being capable of taking over the combined actions of amphiphile and p47 phox , to serve as a carrier for the translocation of p67 phox to the membrane.
Our results support the following model of oxidase assembly. Binding of p67 phox to cytochrome b 559 , via an interaction with either gp91 phox or p22 phox or with both subunits, is responsible for the induction of a conformational change in gp91 phox and the consequent initiation of electron transport leading to O 2 .
generation. The establishment of p67 phox -cytochrome b 559 contact(s) is dependent on translocation of p67 phox from cytosol to the plasma membrane environment; this first, essentially nonspecific, step is followed by movement of p67 phox in the plane of the membrane, culminating in the formation of specific proteinprotein interactions with the cytochrome. Unlike p47 phox , p67 phox is incapable of unassisted translocation to the membrane and, in the intact cell, requires the participation of both p47 phox and Rac, acting as carrier proteins. It is not known whether the interaction of Rac with p67 phox takes place in the cytosol or only following the insertion of Rac into the membrane. Another important question is whether the affinity of Rac for p67 phox is enhanced by prenylation and/or by membrane attachment of Rac. A recent example of such a situation is the correlation between C-terminal processing of Rac1 and its ability to stimulate phospholipase C-␤ 2 (40). Since preny- FIG. 5. Amphiphile-independent activation of NADPH oxidase by prenylated Rac1 is prevented by agents that compete with the membrane for binding of Rac1. A, Rho GDI inhibits amphiphile-independent oxidase activation in a cell-free system consisting of membrane vesicles (5 nM cytochrome b 559 heme), prenylated Rac1-GTP␥S (300 nM), and p67 phox (300 nM) (filled circles). Rho GDI exerts no inhibitory effect when the cell-free system also contains p47 phox (300 nM) and is activated by LiDS lated Rac exists in the cytosol exclusively as a dimer with Rho GDI, membrane attachment of Rac, in vivo, must be preceded by a process resulting in its complete or at least partial dissociation from Rho GDI. We also have to conclude that the carrier roles of p47 phox and Rac, for p67 phox , are not symmetrical and not mutually interchangeable. This is shown by the ability of (Rac ϩ p67 phox ) to fully support oxidase activation in vitro, whereas (p47 phox ϩ p67 phox ) is unable to do so, both in the presence and absence of amphiphile.
The cell-free system described in this report is clearly not an accurate reflection of the in vivo reality, since p47 phox is required for O 2 . production in intact phagocytes. It does, however, provide new information on the molecular mechanisms of oxidase assembly. Thus, the principal, if not the only, role of Rac appears to be to recruit p67 phox to the membrane in a manner that leads to a "productive" interaction with cytochrome b 559 . It is suggested that this recruitment is based exclusively on the ability of prenylated Rac to act as a carrier for p67 phox . However, our data do not exclude the possibility that Rac also interacts with one or both subunits of cytochrome b 559 and that such interaction either generates a novel binding site for p67 phox on cytochrome b 559 or contributes directly to the induction of a conformational change in gp91 phox and to the initiation of electron flow.