Cellubrevin-targeted Fluorescence Uncovers Heterogeneity in the Recycling Endosomes

Abstract

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 transferrinenriched 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 A 1 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.

Organic Anion Transporter OAT1 Undergoes Constitutive and Protein Kinase C-regulated Trafficking through a Dynamin- and Clathrin-dependent Pathway

“…In addition, the partial colocalization of hOAT1 with transferrin and EEA1 could also arise from other possibilities. It has been shown that recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values (51). Therefore, it may be possible that some of hOAT1 resides in a different subpopulation of recycling endosomes from that enriched in transferrin.…”

The Dopamine Transporter Constitutively Internalizes and Recycles in a Protein Kinase C-regulated Manner in Stably Transfected PC12 Cell Lines

“…Surprisingly, we observed a trend toward increased DAT surface expression following bafilomycin treatment, suggesting that early endocytic trafficking of DAT and TfR are mechanistically distinct. DAT may reside in a subpopulation of early endosomes that is relatively insensitive to bafilomycin and/or pH changes such as reported for cellubrevin (52). Alternatively, DAT may be intrinsically less dependent upon pH for endosomal sorting.…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…29,77 Currently, immunoisolation studies are also in progress in an attempt to understand the dynamic regulation of recycling, transcytosis, and the caveolin cycle. However, in view of the complexity of the recycling endosomal compartment it is presumable that the physical separation of the 3 functional subcompartments from RRC will be complicated, 37,45,78 even questionable, considering the intricacy of tubulo-network membrane structures and the molecular mix up in single clathrin-coated pits or caveolae plasma membrane microdomains.…”