Isolation and Characterization of Mutant CHO Cell Lines with Compartment-Specific Resistance to Brefeldin A

• Published in: The Journal of cell biology Vol. 126; no. 1; pp. 65 - 75
• Main Authors: Yan, Jian-Ping, Colon, Manuel E., Beebe, Laurie A., Melançon, Paul
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.07.1994; The Rockefeller University Press
• Subjects: ADP-Ribosylation Factors; Animals; Biological and medical sciences; Brefeldin A; Cell Compartmentation; Cell Division - drug effects; Cell growth; Cell lines; Cell structures and functions; Cells; Cellular biology; CHO cells; CHO Cells - physiology; Coatomer Protein; Cricetinae; Cyclopentanes - metabolism; Cyclopentanes - pharmacology; Dose-Response Relationship, Drug; Drug Resistance - genetics; Endosomes; Fundamental and applied biological sciences. Psychology; Genetic mutation; Golgi apparatus; Golgi Apparatus - drug effects; Golgi Apparatus - ultrastructure; GTP-Binding Proteins - analysis; Hepatocytes; Membrane Glycoproteins; Membrane Proteins - metabolism; Microtubule-Associated Proteins - analysis; Miscellaneous; Models, Biological; Molecular and cellular biology; Mutation; Organelles; Organelles - drug effects; Organelles - ultrastructure; P branes; Phenotype; Selection, Genetic; Viral Envelope Proteins - metabolism; Rodentia; Molecular interaction; Golgi apparatus; Proteins; Resistance; Ovary; Vertebrata; Specificity; Cell line; Mammalia; Mutation; Endosome; Hamster
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.126.1.65

22 CHO BFY (BFY) cell lines were isolated at a frequency 2-30× 10-7 from mutagenized populations on the basis of their ability to grow in the presence of 1 μg/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 μg/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 μg/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle-specific coats.