Wu, Guang; Li, Hai; and Yang, Zhenbiao

Department of Plant Biology and plant Biotechnology Center, The Ohio State University, 1060 Carmack Road, Columbus, OH 43210

Rho-family GTPases control many important processes such as cell morphogenesis, cell division, gene expression, and membrane trafficking in eukaryotes. Three major subgroups of Rho GTPases, CDC42, Rac, and Rho, are found in animals. Our previous studies indicate that a plant-specific subgroup of Rho GTPases termed Rop plays an important role in signal transduction such as in the regulation of polarized cell growth in a calcium dependent manner. However, little is known about other components in Rop-dependent signaling pathways and the connection between the Rop and calcium. In this study, we identified a novel class of Rho GTPase-activating proteins (GAPs) termed RopGAPs that specifically interact with the GTP-bound Rop. An in vivo functional association between Rop GTPases and RopGAPs was suggested by our observation that a green fluorescence protein-tagged RopGAP and a Rop GTPase are both localized to the membrane of endosome-like organelles in tobacco cells. Further study shows that the pollen tube growth of the transgenic Arabidopsis with the overexpression of RopGAPs was inhibited in a calcium dependent manner, suggesting that RopGAPs may directly be involved in the calcium regulation and the polarized cell growth. RopGAPs have a unique structural feature, i.e., the presence of a Cdc42/Rac-interactive binding (CRIB) motif in addition to a conserved Rho GAP domain. A truncated RopGAP1 mutant lacking the CRIB motif has the same GAP activity on Rop1At and a human Cdc42. Compared to this mutant, however, the full-length RopGAP1 has at least 5- fold higher GAP activity on Rop1At, but has 2-fold lower GAP activity on Cdc42. Like the full-length RopGAP1, the GAP domain specifically interacts with the GTP-bound Rop1At, whereas the CRIB motif-containing domain lacking the GAP domain interacts with both GTP-bound and GDP-bound forms of Rop1At. The mutations inside the CRIB domain that disrupt its interaction of Rops cause the RopGAP to lose its GTPase activating activity to the level of a truncated RopGAP1 mutant lacking the CRIB motif. These results indicate a novel CRIB motif-dependent regulatory mechanism by which RopGAPs specifically activate Rop GTPase. This is the first study that demonstrates the existence of a regulatory domain for the GAP activity of GAP within a GTPase-activating protein. We propose that the CRIB motif function as a Rop-specific GAP activator by enhancing the interaction of RopGAPs with the GTP-GDP transitional state of Rop GTPases.