Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells.
We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.
Hansen
S H
SH
Department of Anatomy, Panum Institute, University of Copenhagen, Denmark.
Petersen
O W
OW
Sandvig
K
K
Olsnes
S
S
van Deurs
B
B
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis; D001943:Breast Neoplasms; D002460:Cell Line; D004705:Endocytosis; D005260:Female; D005293:Ferritins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism*; D008854:Microscopy, Electron; D018396:Mucin-1; D012276:Ricin / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D008562:Membrane Glycoproteins; D018396:Mucin-1; D005293:Ferritins; D012276:Ricin; D006735:Horseradish Peroxidase