A Chinese Hamster Ovary Cell Mutant with a Heat-Sensitive, Conditional-Lethal Defect in Vacuolar Function

• Published in: The Journal of cell biology Vol. 99; no. 6; pp. 1907 - 1916
• Main Authors: Marnell, Mary H., Mathis, Linda S., Stookey, Margaret, Shia, Shang-Pwu, Stone, Dennis K., Draper, Rockford K.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.12.1984; The Rockefeller University Press
• Subjects: Acidification; Animals; Biological and medical sciences; Cell Division - drug effects; Cell growth; Cell Line; Cell physiology; Cells; CHO cells; Cricetinae; Cricetulus; Cultured cells; Cytosol; Diphtheria; Diphtheria Toxin - pharmacology; Endocytosis; Female; Fundamental and applied biological sciences. Psychology; Hot Temperature; Lectins - pharmacology; Lesions; Molecular and cellular biology; Mutation; Organoids - ultrastructure; Ovary; Plant Lectins; Ribosome Inactivating Proteins, Type 2; Toxins; Toxins, Biological - pharmacology; Vacuoles - drug effects; Vacuoles - ultrastructure; Pseudomonadales; Cell culture; Acidification; Corynebacterium diphtheriae; Vacuole; Toxin; Endocytosis; Sensitivity resistance; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa; Actinomycetes; Corynebacteriaceae; Cell line CHO; Temperature sensitive mutation
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.99.6.1907

We describe a mutant derived from Chinese hamster ovary cells that is heat-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34°C but does not grow in Dulbecco's modified Eagle's medium at 39.5°C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5°C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5°C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.