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Published 31 March 2003. doi:10.1083/jcb.200211098
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© The Rockefeller University Press, 0021-9525/2003/3/1129 $5.00
The Journal of Cell Biology, Volume 160, Number 7, 1129-1138

Article

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

N.D. Sonawane1,2,3 and A.S. Verkman1,2,3

1 Department of Medicine, University of California, San Francisco, San Francisco, CA 94143
2 Department of Physiology, University of California, San Francisco, San Francisco, CA 94143
3 Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143

Address correspondence to Alan S. Verkman, Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, San Francisco, CA 94143-0521. Tel.: (415) 476-8530. Fax: (415) 665-3847. E-mail: verkman{at}itsa.ucsf.edu

Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, {alpha}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 {alpha}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.

Key Words: endocytosis; transferrin; ratio imaging; chloride channel; cholera toxin

* Abbreviations used in this paper: {alpha}2M, {alpha}2-macroglobulin; BAC, 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate; BAC-dextran, BAC-labeled dextran; BAC-dextran-{alpha}2M-TMR, TMR-{alpha}2M conjugated to BAC-dextran; BAC-dextran-CTb-TMR, TMR-CTb conjugated to BAC-dextran; BAC-dextran-Tf-TMR, TMR-Tf conjugated to BAC-dextran; [Cl-], Cl- concentration; CTb, cholera toxin B subunit; FITC-{alpha}2M-TMR, {alpha}2M-labeled with FITC and TMR; FITC-Tf-TMR, Tf-labeled with FITC and TMR; MBS, 3-(maleimido)benzoic acid succinimidyl ester; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate; Tf, transferrin; TMR, 5- (and 6) carboxytetramethylrhodamine; TMR-{alpha}2M, TMR-labeled {alpha}2M; TMR-Tf, TMR-labeled transferrin.


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