JCB logo
PeproTech: Cell Culture Supplements
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
Published 31 March 2003. doi:10.1083/jcb.200211098
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 340K)
Right arrow PPT slides of all figures
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new content in the JCB
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sonawane, N.D.
Right arrow Articles by Verkman, A.S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sonawane, N.D.
Right arrow Articles by Verkman, A.S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

© 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.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:



  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents