Domain II mutants of Pseudomonas exotoxin deficient in translocation.
Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.
Jinno
Y
Y
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Ogata
M
M
Chaudhary
V K
VK
Willingham
M C
MC
Adhya
S
S
FitzGerald
D
D
Pastan
I
I
D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D001692:Biological Transport; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A