Abstract
Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor.
Highlights
From the Departmentof Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts02139 and §Divisionof Pediatric HematologylOncology, Children’s Hospital MedicaCl enter, Sidney Farber Cancer Institute and Department of Pediatrics, HaruardMedical School, Boston, Massachusetts[021 15]
As a control to ensure that all binding sites/cell, and that the experiment is carried out in of the '"I-labeled cell-associated transferrinis exocytosed the presence of 0.25 mM cycloheximide to block protein synnormally, cells are incubated with'251-ferrotransferrin for 2 h thesis (Ref. 5; data not shown) and, synthesis of at 37 "C; unbound ligand is removed, and the cells are incu- new receptor molecules, we calculate that the time required bated for 25 min at 37 "C in the presenceof 128 nM unlabeled for each receptor molecule to traverse an endocytic cycle is ligand.Cell-associatedradioactivitydeclines to8% of the 1.5 X lo5G9.5 x lo3 = 15.8 min
0.31, only about 30% of ferrotransferrin, recycled to the cell surface bound to its receptor, will be expected to dissociate into the medium; the balance should be re-endocytosed into the cell
Summary
TheFate of TransferrinPolypeptideandIronduring a Single Cycle of Endocytosis-Our first experiments focus on the fateof the protein andiron moieties of transferrin during a single cycle of endocytosis in HepG2cells. The datafor the control experiment (i.e.that remaining on the cell surface) are adapted from Fig. 2, and were recalibrated after subtraction of a background of 6% that represents surface-bound transferrin radioactivity which cannot be rethe bound ferrotransferrin usinagcombination of low pH and desferrioxamine (3, 14, 15).
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