Boujemaa-Paterski R, Gouin E, Hansen G, Samarin S, Le Clainche C, Didry D, Dehoux P, Cossart P, Kocks C, Carlier M F, Pantaloni D
Dynamique du Cytosquelette, LEBS, CNRS, 91198 Gif-sur Yvette, France.
Biochemistry. 2001 Sep 25;40(38):11390-404. doi: 10.1021/bi010486b.
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 (41)DEWEEE(46) and basic (146)KKRRK(150) 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.
基于肌动蛋白的李斯特菌和志贺氏菌的推进作用模拟了运动细胞前沿的向前运动。志贺氏菌利用真核蛋白N-WASp通过Arp2/3复合物刺激肌动蛋白聚合和丝分支，而李斯特菌表面蛋白ActA通过未知机制直接激活Arp2/3复合物。在这里，我们表明ActA的N端结构域以类似于肌动蛋白单体结合蛋白的方式结合一个肌动蛋白单体，并与Arp2/3复合物结合，在催化Arp2/3复合物介导的丝末端分支方面模拟WASp家族蛋白的C端结构域。未发现ActA激活的Arp2/3复合物导致丝侧分支的证据。ActA保守酸性区域（41）DEWEEE（46）和碱性区域（146）KKRRK（150）的突变影响Arp2/3的结合，但不影响G-肌动蛋白的结合。野生型和突变型李斯特菌菌株在活细胞和由纯蛋白重构的培养基中的运动特性证实了生化实验的结论。丝分支后会迅速去分支。当Arp2/3由ActA激活时，去分支速度比由N-WASp的C端结构域激活时快3至4倍。在重构的运动培养基中，VASP是ActA包被的珠子高效推进所必需的，但它不影响ActA激活的Arp2/3介导的丝末端分支/去分支速率，也不影响丝的封端。因此，VASP影响另一个仍未确定的生化反应，该反应在基于肌动蛋白的运动中起重要作用。
