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.

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