Fauré J, Vignais P V, Dagher M C
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Biologie Moléculaire et Structurale, CEA Grenoble, France.
Eur J Biochem. 1999 Jun;262(3):879-89. doi: 10.1046/j.1432-1327.1999.00458.x.
Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [alpha32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.
Rho GTP酶有两种可相互转换的形式和两种细胞定位。在其结合GTP的构象中，它们与细胞膜结合并被激活。在无活性的结合GDP的构象中，它们与一种称为GDP解离抑制剂（GDI）的胞质蛋白结合。我们先前报道，除非该复合物已与磷酸肌醇一起孵育，否则肉毒杆菌C3 ADP-核糖基转移酶无法作用于RhoA/Rho-GDI复合物中的RhoA成分。我们在此表明，磷脂酰肌醇（PtdIns）、磷脂酰肌醇4磷酸（PtdIns4P）、磷脂酰肌醇3,4-二磷酸（PtdIns3,4P2）、磷脂酰肌醇4,5-二磷酸（PtdIns4,5P2）和磷脂酰肌醇3磷酸（PtdInsP3）不仅增强了C3依赖性ADP-核糖基化，还增强了异戊二烯化的RhoA/Rho-GDI复合物中RhoA成分的GDP/GTP交换。相反，在未异戊二烯化的RhoA/Rho-GDI复合物中，ADP-核糖基化和GDP/GTP交换的水平与在游离RhoA上测得的水平相当，并且不受磷酸肌醇的影响。在这两种情况下，磷酸肌醇都使复合物部分打开，但并未使其完全解离。在用磷酸肌醇处理异戊二烯化的RhoA/Rho-GDI复合物后，证实了其向中性粒细胞膜的GTP依赖性转移。使用经PtdIns4P预处理并用[α32P]GTP标记的异戊二烯化RhoA/Rho-GDI复合物进行覆盖分析，发现三种分子量在26至32 kDa之间的膜蛋白被放射性标记。我们得出结论，在磷酸肌醇存在的情况下，异戊二烯化的RhoA/Rho-GDI复合物部分打开，这使得RhoA能够将GDP交换为GTP。打开的GTP-RhoA/Rho-GDI复合物获得了靶向特定膜蛋白的能力。
