Induction of direct endosome to endoplasmic reticulum transport in Chinese hamster ovary (CHO) cells (LdlF) with a temperature-sensitive defect in epsilon-coatomer protein (epsilon-COP).
In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of epsilon-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in epsilon-COP. At the nonpermissive temperature epsilon-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of epsilon-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of epsilon-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of epsilon-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.
Llorente
Alicia
A
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Lauvrak
Silje U
SU
van Deurs
Bo
B
Sandvig
Kirsten
K
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D020755:Coat Protein Complex I / Q000378:metabolism; D020756:Coatomer Protein / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D007457:Iodine Radioisotopes; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012276:Ricin / Q000493:pharmacokinetics / Q000633:toxicity; D013431:Sulfates / Q000378:metabolism
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D007457:Iodine Radioisotopes; D013431:Sulfates; D020126:Brefeldin A; D012276:Ricin; D008358:Mannose