Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling
Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling
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DOI：
10.1074/jbc.274.41.29050
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发表时间：
1999-10-08
影响因子：
4.8
通讯作者：
Just, I
中科院分区：
文献类型：
--
作者：
Genth, H;Aktories, K;Just, I
小 GTP 酶 Rho、Rac 和 Cdc42 在效应器结构域氨基酸苏氨酸 37/35 处被艰难梭菌毒素 A 和 B 进行单糖基化。糖基化通过抑制效应器偶联使 Rho 蛋白失活。为了了解功能后果，分析了糖基化对 Rho GTP 酶在细胞质和细胞膜之间的亚细胞分布和循环的影响。在完整细胞和细胞裂解物中，糖基化导致大部分 RhoA GTP 酶易位至细胞膜，而一小部分在细胞质中呈单体形式，不与鸟嘌呤核苷酸解离抑制剂 (GDI-1) 复合。与 GDI-1 复合的 Rho 不是糖基化的底物，修饰的 Rho 不与 GDI-1 结合。然而，诱导 Rho 从 GDI 复合物中释放的膜因子使得胞质 Rho 可用作糖基化的底物。糖基化 RhoA 与质膜的结合是饱和的，可与未修饰的 Rho-GTP gamma S 鸟苷 5'-O-(3-硫代三磷酸) 竞争，并发生在分子量约为 70 kDa 的膜蛋白处。膜结合的葡萄糖基化 Rho 不能像未修饰的 Rho 那样被 GDI-1 提取，导致修饰的 Rho 在膜结合位点积累。因此，除了效应子偶联抑制之外，糖基化还抑制细胞质和细胞膜之间的 Rho 循环，这是 Rho 激活的先决条件。
The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector domain amino acid threonine 37/35 by Clostridium difficile toxins A and B. Glucosylation renders the Rho proteins inactive by inhibiting effector coupling To understand the functional consequences, effects of glucosylation on subcellular distribution and cycling of Rho GTPases between cytosol and membranes were analyzed. In intact cells and in cell lysates, glucosylation leads to a translocation of the majority of RhoA GTPase to the membranes whereas a minor fraction is monomeric in the cytosol without being complexed with the guanine nucleotide dissociation inhibitor (GDI-1). Rho complexed with GDI-1 is not substrate for glucosylation, and modified Rho does not bind to GDI-1. However, a membranous factor inducing release of Rho from the GDI complex makes cytosolic Rho available as a substrate for glucosylation. The binding of glucosylated RhoA to the plasma membranes is saturable, competable with unmodified Rho-GTP gamma S guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa. Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites. Thus, in addition to effector coupling inhibition, glucosylation also inhibits Rho cycling between cytosol and membranes, a prerequisite for Rho activation.