Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol.
Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na(2)(35)SO(4), the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.
Rapak
A
A
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo.
Falnes
P O
PO
Olsnes
S
S
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012276:Ricin / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells
D012276:Ricin