Listeria protein ActA mimics WASP family proteins: It activates filament barbed end branching by Arp2/3 complex
Listeria protein ActA mimics WASP family proteins: It activates filament barbed end branching by Arp2/3 complex
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DOI：
10.1021/bi010486b
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发表时间：
2001-09-25
期刊：
影响因子：
2.9
通讯作者：
Pantaloni, D
中科院分区：
文献类型：
--
作者：
Boujemaa-Paterski, R;Gouin, E;Pantaloni, D
李斯特菌和志贺氏菌的基于肌动蛋白的推进模仿运动细胞的前缘的向前运动。志贺氏菌利用真核蛋白N-WASp通过Arp 2/3复合物刺激肌动蛋白聚合和细丝分支，而李斯特菌表面蛋白ActA通过未知机制直接激活Arp 2/3复合物。在这里，我们表明，ActA的N-末端结构域结合一个肌动蛋白单体，在一个profilin样的方式，和Arp 2/3复合物和模拟的C-末端结构域的WASp家族蛋白在催化细丝倒刺末端分支Arp 2/3复合物。没有发现ActA激活的Arp 2/3复合物引起的纤维侧支化的证据。ActA的保守酸性（DEWEEE 46）-D-41和碱性（KKRRK 150）-K-146区域的突变影响Arp 2/3结合，但不影响G-肌动蛋白结合。野生型和突变型李斯特菌菌株在活细胞中和在由纯蛋白质重构的培养基中的运动特性证实了生化实验的结论。纤维分支之后是快速脱支。当Arp 2/3被ActA激活时，脱支比被N-WASp的C-末端结构域激活快3-4倍。VASP是在重构的运动介质中有效推进ActA包被的珠粒所必需的，但它不影响ActA活化的Arp 2/3的倒刺末端分支/去分支的速率，也不影响细丝的加帽。因此，VASP影响另一个尚未确定的生化反应，在肌动蛋白为基础的运动中发挥重要作用。
Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (DEWEEE46)-D-41 and basic (KKRRK150)-K-146 regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/ debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.