Determinants of [Cl(−)] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

Chloride concentration ([Cl(−)]) was measured in defined organellar compartments using fluorescently labeled transferrin, α(2)-macroglobulin, and cholera toxin B-subunit conjugated with Cl(−)-sensitive and -insensitive dyes. In pulse-chase experiments, [Cl(−)] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ∼10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl(−)] just after internalization (compared with 137 mM solution [Cl(−)]) was prevented by reducing the interior-negative Donnan potential. [Cl(−)] in α(2)-macroglobulin–labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1–45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl(−) accumulation was prevented by bafilomycin but restored by valinomycin. A Cl(−) channel inhibitor slowed endosomal acidification and Cl(−) accumulation by ∼2.5-fold. [Cl(−)] was 49 mM and pH was 6.42 in cholera toxin B subunit–labeled Golgi complex in Vero cells; Golgi compartment Cl(−) accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl(−) is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl(−)] early after internalization. We propose that reduced [Cl(−)] and volume in early endosomes permits endosomal acidification and [Cl(−)] accumulation without lysis.