Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Mol Biol Cell. 2012 Nov;23(21):4256-72. doi: 10.1091/mbc.E11-12-1022. Epub 2012 Sep 12.
During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.
在网格蛋白介导的胞吞作用中，由 Arp2/3 复合物引发的分支肌动蛋白聚合提供了驱动囊泡内化所需的力。Las17（酵母 WASp）是酵母细胞中 Arp2/3 复合物最强的激活剂；它没有自动抑制作用，并且在肌动蛋白聚合开始前 20 秒到达胞吞作用部位。目前尚不清楚 Las17 如何在胞吞作用部位保持 20 秒的非活性状态，从而将肌动蛋白聚合限制在胞吞作用的后期。在本文中，我们证明 Las17 是与 Sla1 形成的一个大的、生化稳定的复合物的一部分，Sla1 是一种网格蛋白衔接蛋白，可抑制 Las17 的活性。这种相互作用是直接的、多价的和强大的，并映射到新的 Las17 多脯氨酸基序上，这些基序同时属于 I 类和 II 类。体外 pyrene-actin 聚合测定实验确立了 Sla1 对 Las17 活性的抑制依赖于 I 类/II 类 Las17 多脯氨酸基序，并且基于 Sla1 和单体肌动蛋白与 Las17 结合的竞争。此外，活细胞成像显示与 Sla1 的相互作用对于 Las17 正常招募到胞吞作用部位、在最初的 20 秒内抑制以及有效胞吞作用是重要的。这些结果推进了我们对胞吞作用中肌动蛋白聚合调节的理解。
