Phagocytes generate superoxide (O2*-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2*-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox -Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b 559 with three cytosolic components: p47 phox , p67 phox , and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b 559 within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47 phox , the tetratricopeptide repeat and activation domains of p67 phox , and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC 50 ) for and formed a more stable complex (longer half-life) with cytochrome b 559 compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the polybasic stretch at the C terminus of the Rac1 segment; 2) interaction with p22 phox via Trp 193 in the N-terminal SH3 domain of the p47 phox segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67 phox and Rac1 segments complementary to their physical fusion. The PX domain of the p47 phox segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.Phagocytes produce reactive oxygen radicals, part of their microbicidal arsenal, by means of a tightly regulated enzyme complex commonly referred to as NADPH oxidase. At the origin of all oxygen radicals is the superoxide anion (O 2 . ), generated by the NADPH-derived one-electron reduction of molecular oxygen. The O 2 . -generating NADPH oxidase complex (briefly "oxidase") consists of a membrane-associated flavocytochrome (cytochrome b 559 ) comprising two subunits (gp91 phox and p22 phox ) and four cytosolic components (p47 phox , p67 phox , p40 phox , and small GTPase Rac1 or Rac2) (reviewed in Refs. 1-3). Electron flow from NADPH to oxygen occurs along three redox stations, all of which are located on gp91 phox : the NADPH-binding site, FAD, and two non-identical hemes. It is assumed that initiation of electron flow is the consequence of a conformational change in gp91 phox induced by its interaction with p67 phox . The region in p67 phox presumed to be involved in such interaction is known as the "activation domain" and consists of residues 199 -210 (4). It has been suggested that the roles of p47 phox and Rac are to serve as carriers of p67 phox to the membrane or as membrane anchors for p67 phox to enable the correct juxtaposing of the activation domain on p67 phox t...

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , p40 phox , and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)⅐RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)⅐RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)⅐RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)⅐RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)⅐RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)⅐RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67 phox , without the need for an anionic amphiphile, as activator. Both Rac1(GDP)⅐RhoGDI and Rac1(GMPPNP)⅐RhoGDI complexes elicited amphiphileindependent, p67 phox -dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

No abstract

Background: Transport of monoamines into storage vesicles, mediated by the vesicular monoamine transporter 2 (VMAT2), is inhibited by tetrabenazine via an unknown mechanism. Results: We identified residues essential for conformational rearrangements required for tetrabenazine binding and substrate transport. Conclusion: Conformational rearrangements are required for binding of the inhibitor. Significance: The results provide a novel insight into the mechanism of transport.