Isolation and characterization of a brefeldin A-resistant mutant of monkey kidney Vero cells.
Brefeldin A (BFA) is a fungal antibiotic which disrupts protein transport between the endoplasmic reticulum and the Golgi. A BFA-resistant mutant of monkey kidney Vero cells, BER-40, which exhibited about a 90-fold increase in the LD50 of BFA (5.2 ng/ml for Vero cells versus 460 ng/ml for BER-40 cells), has been isolated. The increased resistance of BER-40 cells toward BFA was also manifested in a greatly reduced inhibition of protein secretion by BFA in the mutant and a lack of protection by BFA of the mutant cells from ricin cytotoxicity. Somatic cell hybridization between the Vero and BER-40 cells showed that the BFA-resistance in BER-40 behaved as a codominant trait. The structure of the Golgi region, as examined by immunofluorescence microscopy with antibodies against Golgi markers (the 110-kDa protein and mannosidase II) or with fluorescent lipid NBD-ceramide, was unchanged in the mutant cells as compared to that in the wild-type cells. Treatment of Vero cells with BFA (1 micrograms/ml) or with 2-deoxyglucose plus sodium azide resulted in a rapid release of the 110-kDa protein, mannosidase II, and NBD-ceramide from the Golgi membrane to a more diffuse distribution in the cytosol. In contrast, these three Golgi markers remained to be Golgi-associated following treatment of BER-40 cells with BFA or with 2-deoxyglucose plus sodium azide. Immunoblotting of cell extracts from Vero and BER-40 cells with monoclonal antibody against the 110-kDa protein did not reveal any significant difference in the level of this Golgi marker in the mutant cells. These data suggest that the BFA-resistance mutation in BER-40 has rendered the cyclic pathway of the 110-kDa protein assembly to the Golgi membrane resistant to both BFA and 2-deoxyglucose plus sodium azide.
Chen
C H
CH
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Kuwazuru
Y
Y
Yoshida
T
T
Nambiar
M
M
Wu
H C
HC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology*; D003847:Deoxyglucose / Q000494:pharmacology; D004351:Drug Resistance; D005799:Genes, Dominant; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D008361:Mannosidases / Q000378:metabolism; D009154:Mutation; D011506:Proteins / Q000378:metabolism*; D012276:Ricin / Q000633:toxicity; D019810:Sodium Azide; D014709:Vero Cells / Q000187:drug effects* / Q000378:metabolism
D000935:Antifungal Agents; D001386:Azides; D003517:Cyclopentanes; D011506:Proteins; D020126:Brefeldin A; D000255:Adenosine Triphosphate; D012276:Ricin; D019810:Sodium Azide; D003847:Deoxyglucose; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase