Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol

Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol

Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

Abstract

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 Na₂ ³⁵SO₄, 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.

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: olsnes{at}radium.uio.no.
R. John Collier, Harvard Medical School, Boston, MA
ABBREVIATIONS:

ER,

endoplasmic reticulum;

SLO,

streptolysin O;

PDI,

protein disulfate isomerase;

MBP,

maltose-binding protein;

endo H,

endoglycosidase H;

PNGase F,

endoglycosidase F;

HSP70,

heat shock protein 70;

GRP94,

glucose regulated protein 94