Vilhardt Frederik, van Deurs Bo
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, Copenhagen, Denmark.
EMBO J. 2004 Feb 25;23(4):739-48. doi: 10.1038/sj.emboj.7600066. Epub 2004 Feb 5.
The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 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 b558, 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.
产生超氧化物的吞噬细胞NADPH氧化酶由膜结合的黄素细胞色素b558复合物以及胞质因子p47phox、p67phox和小GTP酶Rac组成，细胞激活后，这些因子转位至膜上以组装活性复合物。我们在此表明，NADPH氧化酶亚基在非离子去污剂TX-100、Brij-58和Brij-98中不溶，是其被纳入富含胆固醇的膜微区（脂筏）的结果。因此，黄素细胞色素b558以胆固醇依赖的方式分离至浮力低密度去污剂抗性膜（DRM）组分，并且胞质NADPH氧化酶因子与低密度DRM动态相关。此外，胆固醇耗竭后完整细胞或无细胞重构系统中的超氧化物产生严重受损，这与胞质phox亚基向膜的转位减少相关。与脂筏作为信号平台的广泛接受的作用类似，我们的数据表明，富含胆固醇的微区在活性NADPH氧化酶的组装中起募集和/或组织胞质NADPH氧化酶因子的作用。
