Plasma membrane localization affects the RhoGAP specificity of Pseudomonas ExoS.
Pseudomonas aeruginosa ExoS (453 amino acids) is a bifunctional type III cytotoxin, comprising a Rho GTPase-activating protein domain (RhoGAP), and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain (termed MLD, residues 51-77) which localizes and traffics ExoS within intoxicated host cells. While membrane localization has been shown to be essential for ExoS to ADP-ribosylate Ras, the relationship between intracellular localization and expression of RhoGAP activity has not been addressed. In this study, loss of MLD function was observed to abolish expression of ExoS RhoGAP activity in HeLa cells. One mutation within the MLD (R56, R63, D70 mutated to N, RRD-->N) diminished plasma membrane localization and altered the cell rounding phenotype elicited by ExoS RhoGAP. In addition, cell rounding caused by ExoS-MLD(RRD-->N) was reversed by dominant active Rac1, but not dominant active Cdc42, indicating a switch in ExoS RhoGAP substrate specificity. Mutation of the C-terminal polybasic region abolished the ability of dominant active Rac1 to protect HeLa cells from expression of the RhoGAP activity of ExoS-MLD(RRD-->N). This study shows the importance of membrane localization in the targeting of Rho GTPases by ExoS RhoGAP.
Zhang
Yue
Y
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
Deng
Qing
Q
Porath
Jaclyn A
JA
Williams
Carol L
CL
Pederson-Gulrud
Kristin J
KJ
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013379:Substrate Specificity
D001427:Bacterial Toxins; D003603:Cytotoxins; D020690:GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D036002:ADP Ribose Transferases; C444820:exoenzyme S