Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol.
Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments. PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain). Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments. Initially, the two fragments were linked by a disulfide bond. Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis. Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment. The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.
Ogata
M
M
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Chaudhary
V K
VK
Pastan
I
I
FitzGerald
D J
DJ
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002460:Cell Line; D004220:Disulfides / Q000032:analysis; D004705:Endocytosis / Q000187:drug effects; D010450:Endopeptidases / Q000378:metabolism*; D005098:Exotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D016296:Mutagenesis; 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; D000643:Ammonium Chloride; D001427:Bacterial Toxins; D004220:Disulfides; D010450:Endopeptidases; D005098:Exotoxins; D008715:Methionine; D008985:Monensin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A