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

A few small GTP-binding proteins of the Ras superfamily have beenidentified in plants, including members of the Rho family: the proteinsbelonging to this group are known in mammalian and yeast cells to beinvolved in the control of polarity, cell morphogenesis and movement byregulating cytoskeleton organization. An investigation into where someRho-like proteins are located in plant cells was made. The antibodies used,anti-Cdc42Hs and anti-Rac 1, were raised against conserved characteristicsequences of Cdc42Hs and Rac1 mammalian proteins respectively. In fixedcells, Cdc42Hs antibody recognized epitopes generally co-localized withmicrotubules which may be implicated in the establishment of cell polarity,whereas the proteins recognized by Rac1 antibody seemed to be associatedwith organelle membranes. The same anti-bodies were used in Western blotsof proteins from tobacco BY-2 and lucerne A2 suspension cells: Cdc42Hsantibody recognized three bands whereas Rac1 antibody revealed only oneband of 18 kDa Mr. A [³⁵S]GTP overlay revealedfour bands of the same Mr as those recognized inWestern blots by Cdc42Hs and Rac1 antibodies.Key words: Rho G-proteins,Cdc42, Rac1, Immunofluorescence, plant cells.