Puertollano Rosa, van der Wel Nicole N, Greene Lois E, Eisenberg Evan, Peters Peter J, Bonifacino Juan S
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development and Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Biol Cell. 2003 Apr;14(4):1545-57. doi: 10.1091/mbc.02-07-0109.
Sorting of transmembrane proteins and their ligands at various compartments of the endocytic and secretory pathways is mediated by selective incorporation into clathrin-coated intermediates. Previous morphological and biochemical studies have shown that these clathrin-coated intermediates consist of spherical vesicles with a diameter of 60-100 nm. Herein, we report the use of fluorescent imaging of live cells to demonstrate the existence of a different type of transport intermediate containing associated clathrin coats. Clathrin and the adaptors GGA1 and adaptor protein-1, labeled with different spectral variants of the green fluorescent protein, are shown to colocalize to the trans-Golgi network and to a population of vesicles and tubules budding from it. These intermediates are highly pleiomorphic and move toward the peripheral cytoplasm for distances of up to 10 microm with average speeds of approximately 1 microm/s. The labeled clathrin and GGA1 cycle on and off membranes with half-times of 10-20 s, independently of vesicle budding. Our observations indicate the existence of a novel type of trans-Golgi network-derived carriers containing associated clathrin, GGA1 and adaptor protein-1 that are larger than conventional clathrin-coated vesicles, and that undergo long-range translocation in the cytoplasm before losing their coats.
跨膜蛋白及其配体在胞吞和分泌途径的各个区室中的分选是通过选择性地掺入网格蛋白包被的中间体来介导的。先前的形态学和生化研究表明，这些网格蛋白包被的中间体由直径为60 - 100 nm的球形囊泡组成。在此，我们报告利用活细胞荧光成像来证明存在一种含有相关网格蛋白包被的不同类型的转运中间体。用绿色荧光蛋白的不同光谱变体标记的网格蛋白以及衔接蛋白GGA1和衔接蛋白-1，显示它们共定位于反式高尔基体网络以及从其出芽的一群囊泡和小管上。这些中间体高度多形，朝着外周细胞质移动，移动距离可达10微米，平均速度约为1微米/秒。标记的网格蛋白和GGA1以10 - 20秒的半衰期在膜上循环进出，与囊泡出芽无关。我们的观察结果表明存在一种新型的源自反式高尔基体网络的载体，其含有相关的网格蛋白、GGA1和衔接蛋白-1，比传统的网格蛋白包被囊泡更大，并且在失去包被之前在细胞质中进行长距离转运。
