Michelot Alphée, Costanzo Michael, Sarkeshik Ali, Boone Charles, Yates John R, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.
Curr Biol. 2010 Nov 9;20(21):1890-9. doi: 10.1016/j.cub.2010.10.016. Epub 2010 Oct 28.
Clathrin-actin-mediated endocytosis in yeast involves the progressive assembly of at least 60 different proteins at cortical sites. More than half of these proteins are involved in the assembly of a branched network of actin filaments to provide the forces required for plasma membrane invagination.
To gain insights into the regulation of endocytic actin patch dynamics, we developed an in vitro actin assembly assay using microbeads functionalized with the nucleation promoting factor (NPF) Las17 (yeast WASP). When incubated in a yeast extract, these beads assembled actin networks, and a significant fraction became motile. Multidimensional protein identification technology (MudPIT) showed that the recruitment of actin-binding proteins to these Las17-derived actin networks is selective. None of the proteins known to exclusively regulate the in vivo formation of actin cables or the actin contractile ring were identified. Our analysis also identified components of three other cortical structures, eisosomes, phosphoinositide kinase (PIK) patches, and the TORC2 complex, establishing intriguing biochemical connections between four different yeast cortical complexes. Finally, we identified Aim3 as a regulator of actin dynamics at endocytic sites.
WASP is sufficient to trigger assembly of actin networks composed selectively of actin patch proteins. These experiments establish that the protein composition of different F-actin structures is determined by the protein factor that initiates the network. The identification of binding partners revealed new biochemical connections between WASP-derived networks and other cortical complexes and identified Aim3 as a novel regulator of the endocytic actin patch.
酵母中网格蛋白-肌动蛋白介导的内吞作用涉及至少60种不同蛋白质在皮质位点的逐步组装。其中一半以上的蛋白质参与肌动蛋白丝分支网络的组装，以提供质膜内陷所需的力。
为深入了解内吞肌动蛋白斑动力学的调控机制，我们开发了一种体外肌动蛋白组装测定法，使用经成核促进因子（NPF）Las17（酵母WASP）功能化的微珠。将这些微珠在酵母提取物中孵育时，它们会组装肌动蛋白网络，并且很大一部分会变得可移动。多维蛋白质鉴定技术（MudPIT）表明，肌动蛋白结合蛋白向这些源自Las17的肌动蛋白网络的募集具有选择性。未鉴定出已知专门调节体内肌动蛋白电缆或肌动蛋白收缩环形成的蛋白质。我们的分析还鉴定出了其他三种皮质结构的成分，即内质体、磷酸肌醇激酶（PIK）斑和TORC2复合物，在四种不同的酵母皮质复合物之间建立了有趣的生化联系。最后，我们鉴定出Aim3是内吞位点肌动蛋白动力学的调节因子。
WASP足以触发由肌动蛋白斑蛋白选择性组成的肌动蛋白网络的组装。这些实验表明，不同F-肌动蛋白结构的蛋白质组成由启动网络的蛋白质因子决定。结合伙伴的鉴定揭示了源自WASP的网络与其他皮质复合物之间新的生化联系，并鉴定出Aim3是内吞肌动蛋白斑的新型调节因子。
