Brown C M, Petersen N O
Chemistry Department, University of Western Ontario, London, Canada.
Biochem Cell Biol. 1999;77(5):439-48.
In this study image correlation spectroscopy was used to demonstrate the presence of two populations of clathrin in situ, on intact cells. In the periphery of the cell approximately 35% of the clathrin triskelions are free within the cytosol while approximately 65% are in large aggregates, presumably coated pits. Although endocytosis is inhibited at low temperature, free clathrin triskelions are still present and small AP-2 aggregates (of approximately 20 proteins), or coated pit nucleation sites, are still observed. Following hypertonic treatment, or cytoplasmic acidification, free clathrin triskelions within the cytosol are depleted and all of the clathrin becomes associated with the membrane. Under these conditions coated pit associated AP-2 remains while the smaller AP-2 aggregates, or coated pit nucleation sites, dissociate. This indicates that the stabilization of AP-2 coated pit nucleation sites requires the presence of free clathrin triskelions within the cytosol. Furthermore, this indicates that free clathrin is required for the early stages of coated pit formation and presumably the continuation of the clathrin-mediated endocytic process. We also provide indirect evidence that AP-2 binding to the membrane in coated pit nucleation sites may be regulated in part by binding to internalization-competent membrane receptors.
在本研究中，图像相关光谱法被用于在完整细胞上原位证明存在两类网格蛋白。在细胞周边，约35%的网格蛋白三脚复合体游离于胞质溶胶中，而约65%存在于大聚集体中，推测为被膜小窝。尽管在低温下内吞作用受到抑制，但游离的网格蛋白三脚复合体仍然存在，并且仍然可以观察到小的AP-2聚集体（约20个蛋白质）或被膜小窝成核位点。在高渗处理或细胞质酸化后，胞质溶胶中的游离网格蛋白三脚复合体减少，所有网格蛋白都与膜结合。在这些条件下，与被膜小窝相关的AP-2仍然存在，而较小的AP-2聚集体或被膜小窝成核位点则解离。这表明AP-2被膜小窝成核位点的稳定需要胞质溶胶中存在游离的网格蛋白三脚复合体。此外，这表明游离网格蛋白是被膜小窝形成早期阶段所必需的，并且推测是网格蛋白介导的内吞过程持续进行所必需的。我们还提供了间接证据，表明在被膜小窝成核位点中，AP-2与膜的结合可能部分受与具有内化能力的膜受体结合的调节。
