A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.
The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b(559), a membrane-associated heterodimer composed of two subunits (Nox2 and p22(phox)), and four cytosolic proteins (p47(phox), p67(phox), Rac, and p40(phox)). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b(559) and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47(phox), p67(phox), and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47(phox)-p67(phox)-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47(phox) phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp(193) in p47(phox) persists. Prenylated GFP-p47(phox)-p67(phox)-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.
Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Ariel
A
Mizrahi
Yevgeny
Y
Berdichevsky
Patrick J
PJ
Casey
Edgar
E
Pick
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017368:Protein Prenylation / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D010743:Phospholipids; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020830:rac1 GTP-Binding Protein