R. Desrosiers, France Gauthier, Julie Lanthier
May 19, 2000
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The Journal of Biological Chemistry
Abstract
The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins,N-acetyl-S-all-trans,trans-farnesyl-l-cysteine andN-acetyl-S-all-trans-geranylgeranyl-l-cysteine, wereused to analyze the role of prenylation in their membrane association. Silver-stained gels indicated thatN-acetyl-S-all-trans-geranylgeranyl-l-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed thatN-acetyl-S-all-trans-geranylgeranyl-l-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-S-all-trans andtrans-farnesyl-l-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted byN-acetyl-S-all-trans-geranylgeranyl-l-cysteine, whereas Ras, which is farnesylated, was not. Furthermore,N-acetyl-S-all-trans-geranylgeranyl-l-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaPi-2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration ofN-acetyl-S-all-trans-geranylgeranyl-l-cysteine. Membrane treatments with 0.7 m KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes byN-acetyl-S-all-trans-geranylgeranyl-l-cysteine likely occurs via different mechanisms.