An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.
An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.
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DOI：
10.1083/jcb.142.4.923
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发表时间：
1998-08-24
期刊：
影响因子：
--
通讯作者：
Maxfield FR
中科院分区：
文献类型：
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作者：
Ghosh RN;Mallet WG;Soe TT;McGraw TE;Maxfield FR
为了检查TGN 38从细胞表面运输到TGN，用嵌合跨膜蛋白TacTGN 38稳定转染CHO细胞。我们使用荧光和125 I标记的抗Tac IgG和Fab片段来跟踪TacTGN 38的胞内后运输。在稳态下，抗Tac主要在TGN中，但在胞吞作用后不久，其主要在早期内体中。11%的细胞TacTGN 38位于质膜上。结合到TacTGN 38的抗体的运输的动力学分析表明，在短的内吞脉冲后，80%的内化的抗-Tac返回到细胞表面（t1/2 = 9分钟），其余的运输到TGN。当较长的填充脉冲和追逐被用来加载到TGN的抗-Tac，它返回到细胞表面的T 1/2的46分钟。定量共聚焦显微镜分析还表明，荧光抗-Tac填充TGN的46分钟的T 1/2。在一个简单的动力学模型中使用测得的速率常数，我们预测82%的TacTGN 38在TGN中，7%在内涵体中。TacTGN 38缓慢地离开TGN，这解释了它的稳态分布，尽管从细胞表面到TGN的靶向效率低。
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t 1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t 1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t 1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.