Wu Xufeng, Zhao Xiaohong, Puertollano Rosa, Bonifacino Juan S, Eisenberg Evan, Greene Lois E
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Biol Cell. 2003 Feb;14(2):516-28. doi: 10.1091/mbc.e02-06-0353.
We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.
我们之前利用光漂白后的荧光恢复技术证明，质膜上网格蛋白包被小窝中的网格蛋白与胞质溶胶中的游离网格蛋白发生交换，这表明网格蛋白包被小窝是动态结构。我们现在研究了反式高尔基体网络中的网格蛋白以及质膜和反式高尔基体网络中网格蛋白包被小窝内的网格蛋白衔接蛋白AP2和AP1是否也与胞质溶胶中的游离蛋白发生交换。我们发现，当网格蛋白包被囊泡的出芽被阻断而不显著影响网格蛋白包被小窝的结构时，质膜上的网格蛋白和AP2以及反式高尔基体网络中的网格蛋白和AP1都能迅速与胞质溶胶中的游离蛋白发生交换。相比之下，当通过高渗蔗糖或钾离子耗竭在质膜或反式高尔基体网络处阻断网格蛋白包被囊泡的出芽时，这两种情况会显著影响网格蛋白包被小窝的结构，此时网格蛋白的交换被阻断，但质膜上的AP2以及反式高尔基体网络中的AP1和GGA1衔接蛋白仍继续快速交换。我们得出结论，网格蛋白包被小窝是动态结构，网格蛋白和衔接蛋白都能快速交换，并且当网格蛋白的交换被阻断时，衔接蛋白能够独立于网格蛋白进行交换。
