Ghosh R N, Mallet W G, Soe T T, McGraw T E, Maxfield F R
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
J Cell Biol. 1998 Aug 24;142(4):923-36. doi: 10.1083/jcb.142.4.923.
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 (t1/2 = 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 t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. 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.
为了研究TGN38从细胞表面向反式高尔基体网络（TGN）的运输，用嵌合跨膜蛋白TacTGN38稳定转染了中国仓鼠卵巢（CHO）细胞。我们使用荧光和125I标记的抗Tac IgG及Fab片段来追踪TacTGN38的内吞后运输过程。在稳态下，抗Tac主要位于TGN中，但内吞后不久它主要位于早期内体中。细胞内11%的TacTGN38位于质膜上。对与TacTGN38结合的抗体运输进行动力学分析表明，在内吞短脉冲后，80%内化的抗Tac返回细胞表面（半衰期=9分钟），其余的运输到TGN。当使用更长的填充脉冲和追踪来将抗Tac加载到TGN中时，它以46分钟的半衰期返回细胞表面。定量共聚焦显微镜分析还表明，荧光抗Tac以46分钟的半衰期填充TGN。在一个简单的动力学模型中使用测量的速率常数，我们预测82%的TacTGN38位于TGN中，7%位于内体中。TacTGN38从TGN缓慢离开，这解释了尽管从细胞表面到TGN的靶向效率低下，但其稳态分布的原因。
