Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Abstract

The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67 phox complex of the resting oxidase. Here we show that this complex contains one copy of each protein, which assembles to form a heterotrimeric complex. The apparent high molecular weight of this complex, as observed by gel filtration studies, is due to an extended, non-globular shape rather than to the presence of multiple copies of any of the proteins. Isothermal titration calorimetry measurements of the interactions between the individual components of this complex demonstrate that p67 phox is the primary binding partner of p47 phox in the resting state. These findings, in combination with earlier reports, allow us to propose a model for the architecture of the resting complex in which p67 phox acts as the bridging molecule that connects p40 phox and p47 phox .

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…Our results are consistent with the latter hypothesis and indicate that the inhibitory effects of mAb NL7 binding to the 498 EKDVITGLK 506 epitope do not appear to directly affect membrane translocation of p47 phox , p67 phox , or Rac. However, there is evidence that cytosolic oxidase subunits may be translocated en bloc as a complex with p40 phox (36), and therefore dock to Cyt b by multiple contact sites. Therefore, obstruction of a single contact by mAb NL7 may be insufficient to prevent membrane translocation, but could inhibit functional interactions between Cyt b and the cytosolic factors.…”

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…We suggest two mechanisms for the targeting p40 phox to phagosomes during Fc␥R-mediated phagocytosis: 1) p40 phox can bind initially to PI(3)P-enriched early endosomes, followed by their fusion to phagosomes, and/or 2) p40 phox can target directly to phagosomes, where PI(3)P is transiently produced during phagosytosis (Ellson et al, 2001a;Gillooly et al, 2001). Because the cytosolic phox proteins exist as a ternary complex (Kuribayashi et al, 2002;Lapouge et al, 2002), we propose a model for Nox2 complex assembly involving these spatiotemporal factors during the Fc␥R-mediated respiratory burst ( Figure 11): 1) in early stages, before phagosome sealing, p47 phox functions as the predominant carrier, mediating translocation of the ternary complex to newly forming phagosomes, 2) in later stages Model of activation and assembly of Nox2 system during phagocytosis. In the resting state, the ternary complex of p47 phox , p67 phox , and p40 phox is in the cytoplasm.…”

“…Although a recent study reported that p40 phox and p67 phox exist as a heterodimer in the resting state and that the p47 phox -containing ternary complex forms only after cell activation (Brown et al, 2003), most other studies to date indicate that the phox proteins exist as a stable 1:1:1 ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in their dephosphorylated state even without cell stimulation ( Figure 1A; Bolscher et al, 1989;Rotrosen and Leto, 1990;Lapouge et al, 2002;Kuribayashi et al, 2002). We examined ternary complex formation by using GFP-p67 phox coexpressed with p47 phox and p40 phox in COS-7 cells and confirmed that all three proteins exist at least partially in a complex that could be detected by immunoprecipitation with antibodies against any of these components ( Figure 1B).…”

“…The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al, 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al, 1989;Rotrosen and Leto, 1990;Kuribayashi et al, 2002;Lapouge et al, 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al, 1994;Zhao et al, 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.…”

“…The interaction between p47 phox and GFP-p67 phox , but not GFP-p47 phox (⌬SH3), is confirmed in immunoprecipitation experiments ( Figure 6B). These data indicate that both p40 phox and p47 phox can function as adaptors of p67 phox to translocate p67 phox to the early endosome and to the plasma membrane, respectively.Because there are a few studies reporting weak interactions between p47 phox and p40 phox detected in vitro and in the yeast two-hybrid system (Sathyamoorthy et al, 1997;Grizot et al, 2001;Lapouge et al, 2002;Massenet et al, 2005), we examined the effect of this interaction on membrane translocation of p47 phox and p40 phox . Coexpression of p40 phox -IRES2-DsRed2 does not influence the translocation of GFPp47 phox to the plasma membrane in RAW 264.7 cells ( Figure 7A, top).…”