Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells.
ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
K J
KJ
Pederson
S
S
Pal
A J
AJ
Vallis
D W
DW
Frank
J T
JT
Barbieri
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D015153:Blotting, Western; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism / Q000382:microbiology; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D012995:Solubility
D001427:Bacterial Toxins; D020690:GTPase-Activating Proteins; D019913:Nuclear Localization Signals; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D011494:Protein Kinases; D000071677:Histidine Kinase