Winter D, Lechler T, Li R
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts, 02115, USA.
Curr Biol. 1999 May 6;9(9):501-4. doi: 10.1016/s0960-9822(99)80218-8.
The Arp2/3 complex is a highly conserved cytoskeletal component that has been implicated in the nucleation of actin filament assembly. Purified Arp2/3 complex has a low intrinsic actin nucleation activity, leading to the hypothesis that an unidentified cellular activator is required for the function of this complex. We showed previously that mutations in the Arp2/3 complex and in Bee1p/Las17p, a member of the Wiskott-Aldrich syndrome protein(WASP) family, lead to a loss of cortical actin structures (patches) in yeast. Bee1p has also been identified as an essential nucleation factor in the reconstitution of actin patches in vitro. Recently, it was reported that WASP-like proteins might interact directly with the Arp2/3 complex through a conserved carboxy-terminal domain. Here, we have shown that Bee1p and the Arp2/3 complex co-immunoprecipitate when expressed at endogenous levels, and that this interaction requires both the Arc15p and Arc19p subunits of the Arp2/3 complex. Furthermore, the carboxy-terminal domain of Bee1p greatly stimulated the nucleation activity of purified Arp2/3 complex in vitro, suggesting a direct role for WASP-family proteins in the activation of the Arp2/3 complex. Interestingly, deletion of the carboxy-terminal domain of Bee1p neither abolished the localization of the Arp2/3 complex, as had been suggested, nor resulted in a severe defect in cortical actin assembly. These results indicate that the function of Bee1p is not mediated entirely through its interaction with the Arp2/3 complex, and that factors redundant with Bee1p might exist to activate the nucleation activity of the Arp2/3 complex.
Arp2/3复合物是一种高度保守的细胞骨架成分，与肌动蛋白丝组装的成核作用有关。纯化的Arp2/3复合物具有较低的内在肌动蛋白成核活性，这导致人们推测该复合物的功能需要一种未鉴定的细胞激活剂。我们之前表明，Arp2/3复合物以及威斯科特-奥尔德里奇综合征蛋白（WASP）家族成员Bee1p/Las17p中的突变会导致酵母中皮质肌动蛋白结构（斑块）的丧失。Bee1p也被确定为体外重建肌动蛋白斑块时的一种必需成核因子。最近，有报道称WASP样蛋白可能通过保守的羧基末端结构域直接与Arp2/3复合物相互作用。在这里，我们已经表明，当以内源水平表达时，Bee1p和Arp2/3复合物会共同免疫沉淀，并且这种相互作用需要Arp2/3复合物的Arc15p和Arc19p亚基。此外，Bee1p的羧基末端结构域在体外极大地刺激了纯化的Arp2/3复合物的成核活性，这表明WASP家族蛋白在激活Arp2/3复合物中具有直接作用。有趣的是，删除Bee1p的羧基末端结构域既没有像之前所认为的那样消除Arp2/3复合物的定位，也没有导致皮质肌动蛋白组装出现严重缺陷。这些结果表明，Bee1p的功能并非完全通过其与Arp2/3复合物的相互作用来介导，并且可能存在与Bee1p功能冗余的因子来激活Arp2/3复合物的成核活性。
