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J. Biol. Chem., Vol. 258, Issue 16, 9681-9689, 08, 1983
A Ciechanover, AL Schwartz, A Dautry-Varsat and HF Lodish
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.
Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents
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