Molecular cloning and expression of a small GTP-binding protein of the Rop family from mung bean

Abstract

The Rho family, a group of small GTP-binding proteins, consists of Rho, Rac and Cdc42 subfamilies in animals and yeasts, and modulates many cellular processes related to the actin cytoskeleton. According to recent study, ROP, a Rho subfamily, is a distinct subgroup found in plant, where it is also involved in the regulation of the cytoskeleton, especially in growth of the tip of the pollen tube. In this study, a rho-related gene was isolated from a mung bean cDNA library and characterized. This gene contains five conserved regions (G1-G5) including the effector-binding domain of many small GTP-binding proteins, and CAAL motif at the C-terminus, suggesting that it might be localized in the membrane. Based on overall homology in its effector-binding domain to those of the Rho family, and to those of plant ROPs, this gene was named VrRop1. The phylogenetic analysis also confirmed that it is related to the Rho families of various species. The VrROP1 protein appears be a member of a subgroup of the ROP family, which includes AtROP1, OsROP5, AtROP7, AtROP3, AtROP4 based on phylogenetic relationship and differential distribution in tissues, especially preferable expression in roots. The transcript of VrRop1 is about 1000 nt long and this gene is likely to exist as a multigene family. Purified recombinant VrROP1 protein can bind to the guanosine nucleotide and has an intrinsic GTP hydrolysis activity, confirming the VrROP1 protein as a typical small GTP-binding protein. However, VrRop1 did not complement rho3 and cdc42 in yeasts. Although the other yeast RHO genes need to be tested, it is likely that VrRop1 might have unique signaling pathways in plants, different from those in yeasts, including controlling actin cytoskeleton. © 2006 Elsevier Ireland Ltd. All rights reserved.