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 tak e place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain t o contain a tyrosine sulfation site alone or in combination with N-gly cosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of (Na2SO4)-S-35, the modified A chains wer e labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allo ws selective labeling of ricin molecules that have already been transp orted retrograde to this organelle. A chain containing C-terminal N-gl ycosylation sites became core glycosylated, indicating retrograde tran sport to the ER. In part of the toxin molecules, the A chain was relea sed from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that transl ocation takes place after transport of the toxin to the ER.