Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol.
Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.
Wesche
J
J
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Rapak
A
A
Olsnes
S
S
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D008565:Membrane Proteins / Q000276:immunology; D008985:Monensin / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000187:drug effects; D009154:Mutation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D000069816:SEC Translocation Channels; D013431:Sulfates / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014709:Vero Cells
D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D007476:Ionophores; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D013431:Sulfates; C067713:lactacystin; D012276:Ricin; D008985:Monensin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium; D000111:Acetylcysteine