Exoenzyme S (ExoS) ADP-ribosylates multiple low-molecular-mass G-(LMMG-) proteins in vitro. Identification of the in vivo substrate specificity of ExoS has been hindered by its bacterial contact delivery into eukaryotic cells and difficulties in identifying ADP-ribosylated proteins within cells. Two-dimensional electrophoresis comparisons of substrate modifications by ExoS in vitro to that following bacterial translocation into HT-29 epithelial cells identified Ras, Ral, and Rab proteins and Rac1 as in vivo substrates of ExoS ADPRT activity. Cellular fractionation studies identified a relationship between membrane association and efficiency of substrate modification. Moreover, Rac and Cdc42 relocalized to the membrane in response to ExoS. Comparisons of substrate modification to time of exposure to ExoS identified a progression of substrate modification, with Ras, RalA, and Rab5 modified first, followed by RabS and 11, then Rab7 and Rac1. The data support that intrinsic properties of LMMG-proteins and their subcellular localization are determinants of bacterially translocated ExoS substrate selectivity. (C) 2002 Elsevier Science (USA).