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J Biol Chem, Vol. 273, Issue 31, 19625-19633, July 31, 1998

Cellubrevin-targeted Fluorescence Uncovers Heterogeneity in the Recycling Endosomes

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200

The pH and trafficking of recycling endosomes have previously been studied using transferrin. We have used another approach, one in which the vesicle transport protein cellubrevin was appended with a luminal IgG epitope to allow targeting of fluorescein-5'-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies to the recycling endosomes in living cells. FITC-F(ab) was specifically internalized by COS cells transfected with cellubrevin-Ig, which at steady state accumulated in a pericentriolar region similar to rhodamine-transferrin. Confocal microscopic analysis showed that endosome labeling by these two markers was heterogeneous. This differential distribution was not induced by the IgG tag, since endogenous Cb and Tf were also partitioned into separate endosomal populations. We used fluorescence ratio imaging of internalized FITC-F(ab) to measure the pH of cellubrevin-enriched recycling endosomes (pH_Cb) and FITC-transferrin to measure the pH of transferrin-enriched recycling endosomes (pH_Tf). In COS cells, cellubrevin endosomes (mean pH_Cb 6.1 ± 0.05; range, 5.2-6.6) were more acidic than transferrin endosomes (mean pH_Tf 6.5 ± 0.05; range, 5.6-7.2). Similar results were obtained in Chinese hamster ovary cells. Treatment with the vacuolar H⁺-ATPase inhibitor bafilomycin A₁ caused pH_Tf to increase (pH_Tf = 1.2 pH units) to a greater extent than pH_Cb (pH_Cb = 0.5 pH units). Furthermore, inhibition of the Na⁺/K⁺-ATPase by ouabain or acetylstrophanthidin caused pH_Tf to decrease by 0.6 pH units but had no effect on pH_Cb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values.

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