NDLI: A Prenylated p47 phox -p67 phox -Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

Content Provider	PubMed Central
Author	Mizrahi, Ariel Berdichevsky, Yevgeny Casey, Patrick J. Pick, Edgar
Abstract	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 Trp193 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.
Related Links	http://dx.doi.org/10.1074/jbc.m110.113779
Ending Page	25499
Page Count	15
Starting Page	25485
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	33
Volume Number	285
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2010-08-13
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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