A Prenylated p67 phox -Rac1 Chimera Elicits NADPH-dependent Superoxide Production by Phagocyte Membranes in the Absence of an Activator and of p47 phox

Activation of the superoxide-generating NADPH oxidase of phagocytes is the result of the assembly of a membrane-localized flavocytochrome (cytochrome b 559 ) with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Activation can be reproduced in an in vitro system in which cytochrome b 559 -containing membranes are mixed with cytosolic components in the presence of an anionic amphiphile. We proposed that the essential event in activation is the interaction between p67 phox and cytochrome b 559 and that Rac and p47 phox serve as carriers for p67 phox to the membrane. When prenylated, Rac can fulfill the carrier function by itself, supporting oxidase activation by p67 phox in the absence of p47 phox and amphiphile. We now show that a single chimeric protein, consisting of residues 1–212 of p67 phox and full-length Rac1 (residues 1–192), prenylated in vitro and exchanged to GTP, becomes a potent oxidase activator in the absence of any other component or stimulus. Oxidase activation by prenylated chimera p67

Superoxide (O 2 . ) is generated by the NADPH-derived oneelectron reduction of molecular oxygen by a plasma membranelocalized flavocytochrome (cytochrome b 559 ) composed of two subunits, gp91 phox and p22 phox (reviewed in Refs. 1 and 2). The catalytic core of the enzyme is gp91 phox , which contains all redox stations through which electrons flow from NADPH to oxygen (3)(4)(5). Stimuli causing O 2 . production activate an intracellular signaling process leading to the initiation of electron transport and O 2 . production. The most likely mechanistic model for this is the induction of a conformational change in gp91 phox , brought about by interaction of cytochrome b 559 with one or more cytosolic components, subsequent to their translocation to the plasma membrane. These components are p47 phox , p67 phox , p40 phox , and the small GTPase Rac (Rac1 or 2). Upon transfer to the membrane, a multi-component complex is assembled, known as the O 2 . -forming NADPH oxidase complex (referred to as "oxidase") (reviewed in Ref. 6). Oxidase assembly can be induced in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac, exposed to an anionic amphiphile serving as activator (7,8). Identifying the cytosolic component(s) responsible for causing the conformational change in gp91 phox is essential for elucidating the molecular basis of this change. The best established fact is the binding of p47 phox to p22 phox , mediated by the interaction between a Src homology 3 domain in p47 phox and a proline-rich region in the C terminus of p22 phox , consequent to the opening of intramolecular bonds in p47 phox (9 -12). Findings from three laboratories demonstrated that, under certain conditions, oxidase activation in vitro is possible in the absence of p47 phox but not in the absence of either p67 phox or Rac (13)(14)(15). This is compatible with a model in which either p67 phox or Rac or both components are responsible for the induction of the conformational change in gp91 phox . A central role for p67 phox is supported by the identification of an "activation domain" in p67 phox consisting of residues 199 -210 (16) or residues 187-193 and 199 -210 (17) that was proposed to interact directly with gp91 phox and regulate electron flow from NADPH to FAD (18). Direct evidence for such a role is offered by the recent description of the binding of p67 phox to whole cytochrome b 559 and to the gp91 phox subunit, an interaction enhanced by the presence of Rac (19). A regulatory effect of Rac on cytochrome b 559 , based on direct interaction between these two components involving the "insert region" of Rac, was also proposed (20) and supported by experimental evidence (21).
We recently put forward a model for oxidase assembly in which a central role is attributed to p67 phox as the only component that makes direct contact with and induces a conformational change in gp91 phox (22). In the intact cell, p67 phox is carried to the membrane habitat of cytochrome b 559 by two carriers, p47 phox and Rac. The "carrier for p67 phox " functions of p47 phox and Rac are not symmetrical, as shown by the fact that oxidase activation in vitro can be achieved by p67 phox ϩ Rac but not by p67 phox ϩ p47 phox (13)(14)(15). To investigate the carrier role of Rac, we made use of the fact that in eukaryotic cells small GTPases undergo posttranslational modification, expressed in part in C-terminal prenylation. Thus, the physiological form of Rac in phagocytes is a protein geranylgeranylated (prenylated) at residue Cys 189 (23). Prenylated Rac1 was found capable of eliciting oxidase activation in a cell-free system containing phagocyte membranes and p67 phox , in the absence of p47 phox and of an activating amphiphile (22), demonstrating that membrane association of Rac was not only required (24) but also sufficient for the induction of oxidase activation.
Recently, this issue was approached by the use of a novel experimental tool, the construction of chimeric proteins consisting of segments of p67 phox and Rac1 (25,26). A chimera consisting of p67 phox truncated at residue 212, fused to fulllength Rac1 and exchanged to GTP␥S, 1 was found capable of amphiphile-dependent oxidase activation in the absence of p47 phox (26). This recombinant chimeric protein was expressed in Escherichia coli and, consequently, nonprenylated. Because Rac in mammalian phagocytes is prenylated and prenylated Rac has a superior ability to associate with membranes, we constructed a series of prenylated p67 phox -Rac1 chimeras. We now demonstrate that a p67 phox (1-212)-Rac1 chimera enzymatically prenylated in vitro and exchanged to GTP␥S elicits NADPH dependent O 2 . production by phagocyte membranes in the absence of an activator and of any other oxidase component. This is the first demonstration of the ability of a single protein to elicit oxidase activation in membranes from resting phagocytes in the absence of any stimulus.

EXPERIMENTAL PROCEDURES
Preparation of Phagocyte Membranes and Membrane Vesicles-Membranes were prepared from guinea pig peritoneal macrophages (7) and solubilized by n-octyl-␤-D-glucopyranoside, and membrane vesicles were produced by dialysis against buffer without detergent (27).
Preparation of Recombinant Oxidase Components-p47 phox and p67 phox were prepared in baculovirus-infected Sf9 cells, and nonprenylated Rac1 was produced in E. coli, by procedures described before (14). p67 phox truncated at residue 212 was produced in E. coli, as described previously (22).
Generation of Nonprenylated p67 phox -Rac1 Chimeras-Four chimeric constructs were generated, joining residues 1-212 of p67 phox with fulllength Rac1 (residues  or parts of it, as recently reported (26). The proteins were expressed in E. coli as glutathione S-transferase fusion proteins and purified by batch affinity chromatography on glutathione-agarose and thrombin cleavage in situ.
Enzymatic Prenylation of p67 phox-Rac1 Chimeras and Rac1 in Vitro-Nonprenylated chimeras and Rac1 were geranylgeranylated in vitro by recombinant mammalian geranylgeranyltransferase type I. Recombinant human geranylgeranyltransferase type I was produced in Sf9 cells coinfected with baculoviruses encoding the two subunits of the enzyme and purified as described (28). Enzymatic prenylation was performed essentially as described in Ref. 29. A typical reaction mixture contained 10 nmol of recombinant p67 phox -Rac1 chimera, 20 nmol of geranylgeranyl pyrophosphate (Sigma), and 10 units of geranylgeranyltransferase type I (one unit transfers 1 nmol of geranylgeranyl to protein/h) in 1 ml of 50 mM Tris-HCl, pH 7.7, 5 mM MgCl 2 , 50 M ZnCl 2 , 2 mM dithioerythritol, and 10 M GDP. The reaction mixture was incubated for 45 min at 37°C on an orbital shaker at 600 rpm, whereupon n-octyl-␤-Dglucopyranoside was added to a final concentration of 4.375 mM, and the reaction continued for additional 45 min. The mixture was ice-cooled and subjected to sonic disruption in an ultrasonic processor (Vibra Cell, 400 w; Sonics and Materials). At this stage, an aliquot was removed for assessing the degree of prenylation by Triton X-114 partition. To the rest of the reaction mixture, glycerol was added to a final concentration of 20% (v/v), bringing the final concentration of n-octyl-␤-D-glucopyranoside to 3.5 mM.
Assessing the Degree of Prenylation by Phase Separation in Triton X-114 -Each preparation of prenylated protein was examined for the success of prenylation by assessing the relative amounts of chimera partitioning in the detergent and aqueous phases upon temperaturedependent phase separation in Triton X-114, as described in Ref. 30. Aliquots from the two phases, brought to equal volumes, were subjected to SDS-PAGE, and the degree of prenylation was estimated from the relative intensities of the bands present in the two phases. The correlation of prenylation with partitioning in the detergent phase was proven by the absence of material in the detergent phase when chimeras were mock-prenylated in the absence of geranylgeranyltransferase type I or geranylgeranyl pyrophosphate.
Nucleotide Exchange-Chimeras or Rac1 were subjected to nucleotide exchange to GTP␥S or GDP␤S (Roche Molecular Biochemicals) as described before (22).
Activation of NADPH Oxidase in Vitro in the Absence of Amphiphile-Prenylated chimeras were tested for the ability to elicit NADPHdependent O 2 . production upon addition to phagocyte membrane vesicles, essentially as described for nonprenylated chimeras (26) but in the absence of an amphiphilic activator. In all experiments, the membranes were present at an equivalent of 5 nM cytochrome b 559 heme, and chimeras were added at concentrations that varied from 50 to 800 nM. When p47 phox was added, it was present at a concentration of 300 nM. This concentration was chosen for the sake of simplicity, based on preliminary experiments showing that activation with 300 mM p47 phox was not different from that using equimolar concentrations of chimera and p47 phox (50 -800 nM, each). The activity of chimeras was compared with that of a combination of isolated p67 phox (1-212) and prenylated Rac1, at concentrations that varied from 50 to 800 nM, each.
Binding of Chimera to Phagocyte Membrane Vesicles-Aliquots of 2 nmol of prenylated chimera were mixed with membrane vesicles (300 pmol equivalent of cytochrome b 559 heme) in a volume of 0.6 ml. The mixtures were kept for 10 min at 4°C and injected in a Superose 12 HR 10/30 fast protein liquid chromatography gel filtration column (Amersham Biosciences). The chimeras were quantified by assessing the functional competence of the p67 phox segment shared by all three chimeras. Thus, membrane-bound and free chimeras were measured by assaying lithium dodecyl sulfate-and NADPH-dependent O 2 . production by 50-l aliquots of column fractions upon supplementation with p47 phox and Rac1-GTP␥S (both at 300 nM), in the absence (membranebound chimera) or presence (free chimera) of membrane vesicles (equivalent of 5 nM cytochrome b 559 heme). The membrane vesicles were quantified by measuring the amount of cytochrome b 559 , as described in Ref. 22.

RESULTS AND DISCUSSION
Generation of Prenylated p67 phox -Rac1 Chimeras-Four nonprenylated p67 phox -Rac1 chimeras were generated in E. coli, as recently described (26), and were subsequently prenylated enzymatically in vitro. The structure and nomenclature of the prenylated chimeras are identical to those of the nonprenylated counterparts and are illustrated in Fig. 1A. Prenylation was confirmed by the partition of over 90% of the product in the detergent layer upon phase separation in Triton X-114 at 37°C (Fig. 1B). The prenylated chimeras had the expected M r , with only minor proteolytic degradation.
Oxidase Activation by Prenylated p67 phox -Rac1 Chimeras-We and others have recently reported that a chimeric protein consisting of the N-terminal residues 1-212 of p67 phox and full-length Rac1 (our chimera 3) is capable of oxidase activation in the presence of an anionic amphiphile, even in the absence of p47 phox (25,26). These chimeras were expressed in E. coli and were therefore nonprenylated. We now demonstrate that the addition of a geranylgeranyl group to the C terminus of chimera 3 results in the formation of a molecule that, when added to phagocyte membranes, elicits dose-dependent NADPHdependent O 2 . generation in the absence of any activator ( Fig.   2A). We examined the activating ability of prenylated chimera 3 exchanged to the nonhydrolyzable nucleotide analog GTP␥S or left in the native state. As seen in Fig. 2A, chimera 3-GTP␥S had a clearly superior activating ability, and the dose-response curve was hyperbolic. The presence of p47 phox added little to the activating capacity of the chimera; V max was 111.6 Ϯ 5.6 mol O 2 . /s/mol cytochrome b 559 heme in the presence of p47 phox and 101.9 Ϯ 5.9, in its absence (means Ϯ S.E. of three experiments, for each group). The corresponding EC 50 values were 145.7 Ϯ 23.9 and 111.7 Ϯ 24.0 nM chimera 3, respectively. The oxidase-activating ability of prenylated chimera 3 in the absence of amphiphile was superior to that of its nonprenylated equivalent assayed in the presence of the amphiphile lithium dodecyl sulfate (26). It also differed from nonprenylated chimera 3 by its almost complete independence from p47 phox . Nonprenylated chimera 3 exchanged to GTP␥S was incapable of activation in the absence of amphiphile, both in the presence and in the absence of p47 phox (Fig. 2A). Amphiphile-independent oxidase activation by prenylated chimera 3-GTP␥S was superior to that elicited by equimolar amounts of the individual fusion partners, namely p67 phox (1-212) and in vitro prenylated Rac1-GTP␥S, whether p47 phox was present or not ( Fig. 2A). The finding that prenylated chimera 3 contains all of the elements required for the induction of electron flow through the redox stations of the oxidase, in the absence of help from another component or activator, is compatible with the proposal that only p67 phox interacts with gp91 phox and that Rac1 functions exclusively as a membrane-targeting molecule (22). To test this proposal, we constructed two additional prenylated p67 phox -Rac1 chimeras. Both contained residues 1-212 of p67 phox , which was fused either to residues 178 -192 of Rac1, containing both the prenylation signal (residues 189 -192) and the polybasic domain (residues 183-188) (chimera 2), or to residues 189 -192 of Rac1, lacking the polybasic domain (chimera 1). Because prenylated chimeras 1 and 2 lack the ability to bind nucleotides, they were assayed in native form, in the absence of amphiphile and in the presence and absence of p47 phox . As apparent in Fig. 2 (B and C), the chimeras were unable to activate the oxidase under conditions in which chimera 3 was markedly active. These results can be interpreted as indicating that the role of Rac is more than just a carrier for p67 phox and that a region upstream of residue 178, missing in chimeras 1 and 2, is required for oxidase activation. The insert region of Rac1 was proposed to play such a role by direct interaction with cytochrome b 559 (20,21). To test this hypothesis, we examined the oxidase-activating ability of a mutant of chimera 3 (chimera 8), in which the insert region of Rac1 (residues 123-133) was deleted. The nonprenylated form of chimera 8 was found to be equal in activity to chimera 3, when tested in the presence of lithium dodecyl sulfate (26). As seen in Fig. 2D, prenylated chimera 8 was capable of oxidase activation in the absence of lithium dodecyl sulfate, although it was less potent than chimera 3, both in the presence and absence of p47 phox . Therefore, removal of the insert region of Rac1 has only a moderate negative effect on the activating capacity of chimera 3, making a direct interaction between the Rac segment of chimera 3 and cytochrome b 559 unlikely.
Exogenous Rac1-GTP␥S Confers Oxidase-activating Ability to Inactive Chimera 2-Another possible reason for the lack of activity of chimeras 1 and 2 is that they are unable to form intramolecular bonds between the p67 phox and Rac1 segments of the chimeras. We proposed (26) the existence of such bonds in the GTP-bound (but not in the GDP-bound) form of chimera 3, and recently direct evidence for this, based on mutating residues involved in p67 phox -Rac1 interaction (31), emerged. 2 Intramolecular bonds were also reported to exist in Ras-Raf1 chimeras, when the Ras segment was in GTP-bound form (32). To test this possibility, we supplemented prenylated chimeras 1 and 2 with equimolar concentrations of nonprenylated Rac1, exchanged to GTP␥S or GDP␤S, and examined their ability to activate the oxidase in the absence of amphiphile and p47 phox . As apparent in Fig. 2B, the addition of Rac1-GTP␥S to the inactive prenylated chimera 2 resulted in substantial oxidase activation; under the same conditions Rac1-GDP␤S was not enhancing. However, supplementing prenylated chimera 1 with Rac1-GTP␥S did not result in enhancement (Fig. 2C). We propose that Rac1-GTP␥S, but not Rac1-GDP␤S, interacts with the p67 phox (1-212) segment of chimeras 1 or 2, forming tripartite complexes (Fig. 3, B and C). Extrachimeric interaction of chimeras 1 and 2 with Rac1-GTP␥S mimics the intrachimeric bonds between the Rac1 and p67 phox segments in prenylated chimera 3 (Fig. 3A). The binding of exogenous Rac1-GTP␥S induces a conformational change in the activation domain of the p67 phox segment, which is essential for a productive interaction with cytochrome b 559 . The Rac-induced conformational change in p67 phox is, so far, hypothetical; no difference in the conformation of free (17) and Rac-associated (31) p67 phox was detected in structural studies, but the latter employed p67 phox truncated at residue 203 and therefore lacked most of the activation domain, including the crucial residue 204 (16). A further condition for oxidase activation is the association of the chimeras with the membrane. The success of chimera 2, as opposed to the failure of chimera 1, to activate in the presence of exogenous Rac1 is best explained by the requirement for two signals for recruitment to the membrane: prenylation and a C-terminal polybasic domain. A "two signals" requirement for membrane localization of prenylated Rac was proposed (24) and demonstrated recently by the use of a Rac1 mutant lacking residues 183-188 (33). The particular roles of Rac and p67 phox in oxidase activation, as revealed by this and an earlier study (26), bear a striking resemblance to those of Ras and Raf1. Thus, it was recently shown that, in addition to its role as a carrier of Raf1 to the membrane, Ras has to interact physically with Raf1 to activate it (34). Prenylated p67 phox -Rac1 Chimeras Associate Spontaneously with Phagocyte Membranes-The ability of prenylated chimera 3 to elicit oxidase activation upon addition to phagocyte membranes in the absence of p47 phox and amphiphile is most readily explained by its spontaneous association with the membrane and the formation of a complex with cytochrome b 559 . We tested this hypothesis directly by quantifying the binding of prenylated chimera 3-GTP␥S and of native chimeras 2 and 1 to phagocyte membrane vesicles by gel filtration on Superose 12. This was based on the finding that membrane vesicles elute in the exclusion volume of the column (7.5 ml), whereas free chimeras elute at 12.9 -14.1 ml. 3 As seen in Fig. 4A, chimeras 3 and 2 bound to membrane vesicles with similar affinity, as shown by their detection in close correlation with the cytochrome b 559 marker; a small amount of chimera 1 was also found associated with the membrane. The mere addition of NADPH to fractions containing membrane-associated chimera 3 led to O 2 . production (Fig. 4B); only minimal O 2 . production by fractions containing membrane-bound chimera 2 and no O 2 .
production by fractions containing the small amount of membrane-bound chimera 1 were detected. These findings are in agreement with the direct assessment of the oxidase-activating ability of the chimeras (Fig. 2), with one exception: membranebound chimera 2 did exhibit a low level of activity in the gel filtration assay that was not detectable in the regular cell-free assay. The results provide further support for our proposal that oxidase activation by prenylated p67 phox -Rac1 chimeras depends on the fulfillment of two conditions: high affinity membrane association resting on two signals and the presence of intrachimeric bonds between the p67 phox and Rac segments of the chimera (Fig. 4, bottom panel). This is, to the best of our knowledge, the first report of a single recombinant protein capable of NADPH oxidase activation in vitro, in the absence of any other oxidase component and stimulus. These results support a model of oxidase activation in which p67 phox acts as the only cytosolic component responsible for inducing a conformational change in the gp91 phox subunit of cytochrome b 559 . Prenylated Rac1 translocates to the membrane and serves either as a carrier or as a membrane anchor for p67 phox . In addition, the interaction of Rac1 with p67 phox results in a conformational change in the activation domain of p67 phox , which is essential for productive interaction with cytochrome b 559 . This model is not to be interpreted as a negation of the role of p47 phox in oxidase activation in vivo. p47 phox is a key participant in the stimulus-elicited oxidase activation in the intact phagocyte, as shown by the impairment of O 2 . production in chronic granulomatous disease patients lacking p47 phox . p47 phox probably functions both as a carrier for p67 phox and as a component interacting directly with gp91 phox (35). It is likely that in the in vivo reality Rac and p67 phox cooperate in bringing about the optimal juxtaposition of the p67 phox activation domain with the corresponding motif on gp91 phox .