Abstract. To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification.
M^Nv viruses and toxins require internalization and exposure to an acidic pH in order to penetrate into the cytosol (8,20,24). A number of cell lines that are defective in endocytosis and/or endocytic acidification have been isolated by taking advantage of this fact. Robbins et al. (6,15,16,19) have described a number of cell lines, selected for diphtheria toxin resistance and impaired mannose-6-phosphate receptor activity, that are resistant to infection by Sindbis virus and vesicular stomatitis virus, as well as protein toxins. Some mutants were found to be defective in the uptake of iron from transferrin, a phenotype linked to their defect in vacuolar acidification, and some exhibited altered Golgi apparatus functions as well. Selection for resistance to both modeccin and diphtheria toxin enabled Draper and co-workers (7, 22) to isolate Chinese hamster ovary cell lines that were temperature sensitive for viability, resistance to protein toxins (diphtheria toxin, modeccin, Pseudomonas exotoxin A), and ATP-stimulated acidification of endocytic vesicles. The temperature-sensitive lesion in viability could be overcome by the addition of FeSO4 to the growth media. Merion et al. (9) have characterized Chinese hamster ovary acidification mutants resistant to Pseudomonas exotoxin A, which were found to be cross-resistant to diphtheria toxin and several animal viruses. Monensin-resistant mutants have been isolated th...
