Differential localization of Rho GTPases in live cells: Regulation by hypervariable regions and RhoGDI binding

Determinants of membrane targeting of Rho proteins were investigated in liv e cells with green fluorescent fusion proteins expressed with or without Rh o-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable r egion determined to which membrane compartment each protein was targeted. T argeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rad, Rad, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plas ma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized I-I-Pas, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rad was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIa. OncogenicDbl stimulated translocation of green fluorescent protein (GFP)-Racl, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to G FP-Cdc42hs was not affected by substituting farnesylation for geranylgerany lation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha bindi ng. Mutations that render RhoA, Cdc42hs, or Rad, either constitutively acti ve or dominant negative abrogated binding to RhoGDIa and redirected express ion to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like thei r functions, are diverse and dynamic.