The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly

Abstract

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b 558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b 558 , in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.

Mouse neutrophils lacking lamin B-receptor expression exhibit aberrant development and lack critical functional responses

“…Studies of a human with homozygous LBR mutations suggested that the cause of Greenberg/HEM dysplasia was due to disrupted sterol Δ 14 reductase activity [16]. Interestingly, differentiated HL-60 cells depleted in cholesterol have a decreased respiratory burst response to both PMA and OZ, and show defective cell shape changes in response to a chemotactic peptide [48,49]. Thus a loss of sterol Δ 14 reductase activity caused by the ic mutation may contribute to the deficient respiratory burst and chemotaxis in ATRA-induced EPRO-ic/ic cells.…”

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages

“…Blockade of CCP internalization by expression of dominant negative EPS15-EH29 increased the cell surface levels of cyt b 558 in Ra2 microglia. A CCP internalization route of cyt b 558 may seem surprising as cyt b 558 on the plasma membrane resides in cholesterol-rich membrane microdomains (lipid rafts) (12), which is sometimes taken to indicate internalization by routes other than the CCP pathway. However, we and others have shown that distribution of membrane proteins in lipid rafts does not preclude endocytosis through coated pits (28,44,45).…”

“…Although superoxide or hydrogen peroxide production is presumed to be highly compartmentalized to achieve anything from pathogen killing to specific redox signaling (11), the sorting receptors and trafficking events that mediate the final subcellular distribution of NOX family members are largely unknown, and no sorting motifs in any NOX family member has been identified. Although NOX family members have been associated by morphological and biochemical techniques with different potential endocytic structures, including lipid rafts (12) and caveolae (8,13), there is no evidence for a role of these trafficking platforms in bulk internalization of NOX family members.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Additionally, NOX-mediated effects on e.g. cell proliferation or migration also depend on strict spatial localization of NOX activation in association with the cytoskeleton (70,71) or in membrane rafts (43,72,73), and on direct interactions with target proteins (3,43,55). This allows for effective oxidative signaling in the presence of abundant cytosolic antioxidant mechanisms (Cu/Zn SOD, GSH peroxidase), and assures confined oxidant production and target specificity.…”