A Chinese hamster ovary cell mutant with a heat-sensitive, conditional-lethal defect in vacuolar function.
We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees 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 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004167:Diphtheria Toxin / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D037102:Lectins / Q000494:pharmacology; D009154:Mutation; D009940:Organoids / Q000648:ultrastructure*; D010053:Ovary; D037121:Plant Lectins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects / Q000648:ultrastructure*
D004167:Diphtheria Toxin; D037102:Lectins; D037121:Plant Lectins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C016004:modeccin