Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly
Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly
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DOI：
10.1074/jbc.m006013200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Pick, E
中科院分区：
文献类型：
--
作者：
Gorzalczany, Y;Sigal, N;Pick, E
超氧化物（O-2）吞噬细胞产生NADPH氧化酶复合体由膜相关黄细胞色素（细胞色素b559）和四个细胞质蛋白p47(phox) p67(phox) p40（phox）和小GTPase Rac （Rac1或-2）组成。NADPH氧化酶的激活（O-2的产生）是部分或全部细胞质成分与细胞色素b组装的结果（559）。这个过程可以在一个由吞噬细胞膜、p47(phox) p67（phox）和Rac1组成的体外系统中复制，Rac1由阴离子两亲体激活。我们现在表明，翻译后加工（戊烯基化）的Rad在含有吞噬膜囊泡和p67（phox）的无细胞系统中启动NADPH氧化酶组装，表达为O-2，在没有激活的两亲体和p47（phox）的情况下产生。丙烯化Cdc42Hs是一种与Rac密切相关的GTPase，在相同的条件下是无活性的。用部分纯化的细胞色素b（559）替代吞噬膜囊泡获得的结果可以完全复制，细胞色素b（559）掺入磷脂酰胆碱囊泡中。经戊烯基化而非非戊烯基化的Rac1可以自发地与吞噬细胞膜囊泡和人工的、无蛋白的磷脂酰胆碱囊泡结合，这一过程被Rho的GDP解离抑制剂抵消。烯丙基化的Rac1与膜囊泡的结合伴随着p67（phox）聚集到相同的位置，并形成一个组装的NADPH氧化酶复合物，在加入NADPH后产生O-2。Rho GDP解离抑制剂和磷脂酰胆碱囊泡抑制两亲性和p47（phox）非依赖性NADPH氧化酶的激活，两者都与膜竞争丙烯酰化的Rac1。我们得出结论，在体外，将Rac靶向吞噬细胞膜足以诱导NADPH氧化酶组装，这表明Rac的主要或可能是唯一作用是将细胞质p67（phox）招募到膜环境中，然后是p67（phox）与细胞色素b的相互作用（559）。
The superoxide (O-2(.-))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b559) and four cytosolic proteins, p47(phox) p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O-2 production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox) p67(phox), and Rac1 activated by an anionic amphiphile. We now show that posttranslationally processed (prenylated) Rad initiates NADPH oxidase assembly, expressed in O-2, production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O-2 upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rad is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).