Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here, we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below the lattice. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the coat. We propose that this design enables an efficient and regulated function of the endocytic coat during endocytic vesicle formation.

中文翻译：

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通过FRET显微镜分析的内吞层的蛋白质结构。

胞吞作用是基本的细胞运输途径，其需要多蛋白内吞涂层的有组织的组装以将质膜拉入细胞。尽管内吞层的蛋白质组成是已知的，但其功能结构尚不十分清楚。在这里，我们通过酵母菌酿酒酵母中的FRET显微镜确定内吞层的纳米级组织。我们评估了18个保守的与外套相关的蛋白质的成对邻近性，并使用网格蛋白亚基和蛋白质截短作为分子标尺来获得外套的高分辨率蛋白质图。此外，我们跟踪了膜内陷过程中外壳蛋白的重排及其在胞吞位点的结合动力学。我们发现，内吞外壳蛋白并不局限于网格蛋白晶格内，但在晶格的上方和下方形成不同的功能层。重要的是，关键的内吞蛋白横穿网格蛋白晶格深深地进入细胞质，从而连接了被膜的膜和细胞质部分。我们建议这种设计使内吞囊泡形成过程中的内吞涂层的有效和调节功能。