MAPPING OF RAS-RELATED GTP-BINDING PROTEINS BY GTP OVERLAY FOLLOWING 2-DIMENSIONAL GEL-ELECTROPHORESIS

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two -dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recomb inant vaccinia virus expression system. Membrane preparations from fel ts expressing the cDNAs were subjected to high-resolution two-dimensio nal polyacrylamide gel electrophoresis followed by [alpha-P-32]GTP lig and blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblot ting with specific antibodies. Rab and Rho proteins cou ld be specifically identified by extraction of streptolysin O-permeabi lized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dis sociation inhibitor. We applied the reference mapping to analyze the G TP-binding patterns of synaptosome fractions from rat brain. The purif ied synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, a nd several other GTPases. This approach and the map we have produced s hould provide a useful aid for the analysis of the expression and loca lization of members of all families of small GTP-binding proteins in v arious cell types and subcellular fractions.