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

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switches are regulated by cycling between GTP- and GDP-bound forms, being activated upon binding of GTP A few small GTP-binding proteins of the Ras superfam- and inactivated when the bound GTP is hydrolysed to ily have been identified in plants, including members GDP by the intrinsic GTPase activity (NuoVer and Balch, of the Rho family: the proteins belonging to this group 1994). These conversions are modulated by interactions are known in mammalian and yeast cells to be involved with cytosolic guanine dinucleotide-dissociation inhibitors in the control of polarity, cell morphogenesis and (GDI ) or stimulators (GDS ), and GTPase activating movement by regulating cytoskeleton organization. An proteins (GAP). However, these regulatory functions are investigation into where some Rho-like proteins are dependent upon prior post-translational modifications of located in plant cells was made. The antibodies used, the carboxy terminus, with isoprenylation, cleavage and anti-Cdc42Hs and anti-Rac1, were raised against con- carboxymethylation (Hall and Zerial, 1995). Among these served characteristic sequences of Cdc42Hs and Rac1 GTPases, four subsets of the Ras superfamily have been mammalian proteins respectively. In fixed cells, identified in plant cells: the Ran proteins which regulate Cdc42Hs antibody recognized epitopes generally co- the transition into M phase (Ach and Gruissem, 1994); localized with microtubules which may be implicated the Arf proteins which may modulate vesicle budding in the establishment of cell polarity, whereas the and coating within the Golgi apparatus through a driver proteins recognized by Rac1 antibody seemed to be cycle (Regad et al., 1993); the Ypt/Rab proteins known associated with organelle membranes. The same anti- to be part of the protein machinery involved in vesicular bodies were used in Western blots of proteins from transport ( Terryn et al., 1993; Hawes and Satiattobacco BY-2 and lucerne A2 suspension cells: Jeunemaitre, 1996; Borg et al., 1997); and the Rho Cdc42Hs antibody recognized three bands whereas proteins ( Yang and Watson, 1993; Delmer et al., 1995; Rac1 antibody revealed only one band of 18 kDa M r .A Lin et al., 1996; Borg et al., 1997; Lin and Yang, 1997). [c35S]GTP overlay revealed four bands of the same M r Most if not all of the studies on Rho in mammalian, as those recognized in Western blots by Cdc42Hs and Drosophila and yeast cells indicate that these proteins Rac1 antibodies. regulate actin organization, especially where the actin is required in cell motility and cell adhesion, although the