Immunodetection of Rho-like plant proteins with Rac1 and Cdc42Hs antibodies

A few small GTP-binding proteins of the Ras superfamily have been identified in plants, including members of the Rho family: the proteins belonging to this group are known in mammalian and yeast cells to be involved in the control of polarity, cell morphogenesis and movement by regulating cytoskeleton organization. An investigation into where some Rho-like proteins are located in plant cells was made. The antibodies used, anti-Cdc42Hs and anti-Raci, were raised against conserved characteristic sequences of Cdc42Hs and Raci mammalian proteins respectively. In fixed cells, Cdc42Hs antibody recognized epitopes generally colocalized with microtubules which may be implicated in the establishment of cell polarity, whereas the proteins recognized by Raci antibody seemed to be associated with organelle membranes. The same antibodies were used in Western blots of proteins from tobacco BY-2 and lucerne A2 suspension cells: Cdc42Hs antibody recognized three bands whereas Raci antibody revealed only one band of 18 kDa Mr. A [γ35S]GTP overlay revealed four bands of the same Mr as those recognized in Western blots by Cdc42Hs and Raci antibodies.