Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
Curr Biol. 2011 Sep 13;21(17):1450-9. doi: 10.1016/j.cub.2011.07.046. Epub 2011 Sep 1.
Genetic analyses of budding and fission yeast identified >50 proteins that assemble at sites of clathrin-mediated endocytosis in structures called actin patches. These proteins include clathrin, clathrin-interacting proteins, actin binding proteins, and peripheral membrane proteins such as F-BAR proteins. Many questions remain regarding the interactions of these proteins, particularly the participation of F-BAR proteins in the assembly of actin filaments.
Our microscopic and genetic interaction experiments on fission yeast show that F-BAR proteins Cdc15p and Bzz1p accumulate in two distinct zones on invaginating membrane tubules and interact with Myo1p and Wsp1p, nucleation-promoting factors for Arp2/3 complex. The two F-BAR proteins peak prior to movement of the actin patch and their accumulation in actin patches depends on the nucleation-promoting factors. At their peak local concentrations, we estimated the stoichiometries of the proteins in actin patches to be one Bzz1p per two Wsp1p and one Cdc15p per Myo1p. Purified Bzz1p has two SH3 domains that interact with Wsp1p and stimulate actin polymerization by Arp2/3 complex. Cells lacking either Cdc15p or Bzz1p assemble 3- to 5-fold less actin in patches (in spite of normal levels of Wsp1p, Myo1p, and Arp2/3 complex), and patches move shorter distances from the plasma membrane.
We propose that during clathrin-mediated endocytosis, F-BAR proteins interact with nucleation-promoting factors to stimulate Arp2/3 complex in two different zones along the invaginating tubule. We further propose that polymerization of actin filaments in these two zones contributes to membrane scission.
芽殖酵母和裂殖酵母的基因分析鉴定出 50 多种蛋白质，这些蛋白质在被称为肌动蛋白斑的结构中，在网格蛋白介导的内吞作用部位组装。这些蛋白质包括网格蛋白、网格蛋白相互作用蛋白、肌动蛋白结合蛋白和质膜外周蛋白，如 F-BAR 蛋白。关于这些蛋白质的相互作用，还有许多问题仍然存在，特别是 F-BAR 蛋白在肌动蛋白丝组装中的参与。
我们对裂殖酵母的显微镜和遗传相互作用实验表明，F-BAR 蛋白 Cdc15p 和 Bzz1p 在向内凹陷的膜小管上积累在两个不同的区域，并与肌球蛋白 1p（Myo1p）和 Wsp1p 相互作用，后者是 Arp2/3 复合物的成核促进因子。这两种 F-BAR 蛋白在肌动蛋白斑移动之前达到峰值，并且它们在肌动蛋白斑中的积累依赖于成核促进因子。在其局部浓度峰值时，我们估计肌动蛋白斑中蛋白质的计量比为每个 Bzz1p 对应两个 Wsp1p 和一个 Cdc15p 对应一个 Myo1p。纯化的 Bzz1p 有两个 SH3 结构域，与 Wsp1p 相互作用，并通过 Arp2/3 复合物刺激肌动蛋白聚合。缺乏 Cdc15p 或 Bzz1p 的细胞在斑中的肌动蛋白组装量减少 3-5 倍（尽管 Wsp1p、Myo1p 和 Arp2/3 复合物的水平正常），并且斑从质膜移动的距离更短。
我们提出，在网格蛋白介导的内吞作用过程中，F-BAR 蛋白与成核促进因子相互作用，以沿向内凹陷的小管在两个不同区域刺激 Arp2/3 复合物。我们进一步提出，这两个区域中肌动蛋白丝的聚合有助于膜分裂。
