Sun Yidi, Leong Nicole T, Wong Tiffany, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Mol Biol Cell. 2015 Nov 1;26(21):3841-56. doi: 10.1091/mbc.E15-04-0252. Epub 2015 Sep 2.
More than 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3 and the CIN85-related protein Sla1 are known to interact with each other in vitro, and they all appear after endocytic-site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here we used live-cell imaging in parallel with genetics and biochemistry to explore comprehensively the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites before Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, whereas the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated, using the auxin-inducible degron system. The results lead us to propose that endocytic-site initiation and actin assembly are separable processes linked by a Pan1/End3/Sla1 complex. Finally, our study provides mechanistic insights into how Pan1 and End3 function with Sla1 to coordinate cargo capture with actin assembly.
60多种高度保守的蛋白质在酵母和哺乳动物中网格蛋白介导的内吞作用位点依次出现。已知酵母中与Eps15相关的蛋白质Pan1和End3以及与CIN85相关的蛋白质Sla1在体外相互作用，它们均在内吞作用位点起始后但在内吞作用肌动蛋白组装之前出现，而肌动蛋白组装有助于膜内陷/切割。在这里，我们结合遗传学和生物化学方法，利用活细胞成像技术全面探究了Pan1、End3和Sla1的动态相互作用及功能。我们的结果表明，Pan1和End3以稳定的方式结合，并在Sla1之前出现在内吞作用位点。End3的C末端通过与Pan1相互作用，对其皮质定位来说是必要且充分的，而End3的N末端在招募Sla1方面起着关键作用。我们使用生长素诱导的降解系统，系统地研究了同时消除Pan1和End3的细胞中内吞蛋白的动态行为。这些结果使我们提出，内吞作用位点的起始和肌动蛋白组装是由Pan1/End3/Sla1复合物连接的可分离过程。最后，我们的研究为Pan1和End3如何与Sla1协同作用以协调货物捕获与肌动蛋白组装提供了机制上的见解。
