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, alpha2-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 approximately 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 alpha2-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 approximately 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.
Sonawane
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ND
Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Verkman
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AS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D018942:Macrolides; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D014634:Valinomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008024:Ligands; D018942:Macrolides; D014634:Valinomycin; D002713:Chlorine; C040929:bafilomycin A1