Bafilomycin A1 Treatment Retards Transferrin Receptor Recycling More than Bulk Membrane Recycling

Abstract

After internalization via clathrin-coated pits, endocytosed material rapidly enters sorting endosomes, organelles that sort recycled molecules such as transferrin (Tf) 1 bound to the transferrin receptor (Tf-R) from lysosomally destined molecules such as low density lipoprotein (1-3). We have provided evidence for a sorting mechanism in which components not carrying special retention or targeting signals are removed from the sorting endosome by a default process based on the geometry of the sorting endosome and an iterative fractionation mechanism (1, 3). Most of the fluid brought into the tubulo-vesicular sorting endosome fills a spherical lumen, whereas most of the surface area is on tubular extensions that are involved in the removal of membrane components from the sorting endosome for export along the recycling pathway (1-3). Tf remains attached to its receptor throughout the recycling pathway, but it is converted to apo-Tf by the low pH of endosomes (4, 5). Occupied Tf-R are transported from sorting endosomes to the endocytic recycling compartment, which in CHO cells is a collection of tubules concentrated near the centriole (6). The Tf-R are returned to the cell surface from the endocytic recycling compartment, and apo-Tf dissociates from the receptor upon return to the surface (5). It has been shown that internalized lipid analogs such as 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexoyl-sphingosylphosphorylcholine (C 6 -NBD-SM) follow the same endocytic recycling pathway as Tf, indicating that in the absence of specialized signals membrane molecules will recycle efficiently from endosomes to the cell surface (3). Endocytosed fluid and ligands released from their receptors by low endosomal pH are retained in the lumen of the sorting endosome and accumulate. The vesicular portions of the sorting endosomes become the vesicles that fuse with late endosomes (7-9).Acidification of endosomes by ATP-dependent proton pumps is important for several aspects of endocytic trafficking. Two well established functions of the low pH of endosomes are to dissociate lysosomally destined ligands from membrane-associated receptors and to strip Fe 3ϩ from transferrin (4, 5, 10, 11). Two additional roles for endosome acidification have been proposed based on studies of drug-treated cells or mutant cell lines, but these are not well understood at present. Inhibition of endosome acidification has been associated with slowed endocytic recycling and intracellular retention of recycling receptors (12)(13)(14). In addition, the delivery of endocytosed molecules to late endosomes or lysosomes is affected by treatments that impair acidification (15, 16). Unfortunately, both of these effects appear to vary among cell lines.Several studies have shown that weak bases and ionophores * This work was supported by National Institutes of Health Grant DK27083 (to F. R. M.) and American Cancer Society Grant CB-8 (to T. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must th...

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