Ciechanover A, Schwartz A L, Dautry-Varsat A, Lodish H F
J Biol Chem. 1983 Aug 25;258(16):9681-9.
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.
生长中的HepG2细胞含有50,000个功能性表面转铁蛋白结合位点（Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267 - 275）和100,000个细胞内位点。在[59Fe]转铁蛋白饱和浓度下，以及在蛋白质合成被阻断的条件下，铁摄取在数小时内呈线性，速率为9,500个转铁蛋白分子/细胞/分钟。因此，每个受体平均每15.8分钟必须循环一次配体。表面结合的转铁蛋白迅速被内吞（t1/2 = 3.5分钟）。所有的铁都保留在细胞内，而脱铁转铁蛋白迅速（t1/2 = 5.0分钟）分泌到培养基中。此前，我们发现（Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258 - 2262），将铁转铁蛋白-受体复合物暴露于pH小于5.0的培养基中会导致铁解离，但脱铁转铁蛋白仍与受体结合。如果将pH提高到7.0，比如当酸性细胞内囊泡与质膜融合时会发生这种情况，脱铁转铁蛋白会非常迅速地（在37℃时t1/2 = 17秒）从其受体上解离。综上所述，这些结果表明转铁蛋白在整个内吞循环中都与受体结合。在本研究中，我们直接测量了转铁蛋白受体循环中涉及的所有动力学参数。它们与同一细胞系中的去唾液酸糖蛋白受体的参数相似，并且可以用一个简单的动力学模型来描述。在存在溶酶体促渗剂的情况下，铁转铁蛋白与其表面受体结合并正常内化。然而，铁不会从转铁蛋白上解离，铁转铁蛋白会循环回到细胞表面并分泌到培养基中。我们得出结论，内吞囊泡中的低pH对于铁从转铁蛋白上解离并传递到细胞中至关重要，但对于转铁蛋白及其受体的循环不是必需的。
