The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation
The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation
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DOI：
10.1074/jbc.m109.054957
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发表时间：
2010-03-12
影响因子：
4.8
通讯作者：
Goode, Bruce L.
中科院分区：
文献类型：
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作者：
Balcer, Heath I.;Daugherty-Clarke, Karen;Goode, Bruce L.
Arp2/3 复合体是一种保守的七亚基肌动蛋白成核机器，由 WASp（Wiskott Aldrich 综合征蛋白）激活。尽管 Arp2/3 复合物在广泛的细胞过程中具有核心重要性，但其机制的许多关键方面尚未得到解决。特别是，复合物中的一些单个亚基尚未被指定明确的功能角色，包括 p40/ARPC1。在这里，我们通过分析针对其保守表面的 39 个整合等位基因，剖析了酿酒酵母 p40/ARPC1（由必需的 ARC40 基因编码）的结构和功能。我们在 p40/ARPC1 上确定了体内功能所需的三个不同位点：一个位点接触 p19/ARPC4，一个位点接触 p15/ARPC5，一个位点位于 p40/ARPC1 的延伸结构“臂”中。使用一种新策略，我们从酵母中纯化了相应的致死突变体 Arp2/3 复合物，并比较了它们的肌动蛋白成核活性。与 p19/ARPC4 接触处的致命突变特异性地损害了 WASp 诱导的成核。相比之下，在没有 WASp 的情况下，与 p15/ARPC5 接触的致命突变导致成核不受调节（“泄漏”）。延伸臂中的致命突变大大减少了成核，并且相同的突变破坏了纯化的 p40/ARPC1 臂结构域结合 WASp 的 VCA 结构域的能力。总之，这些数据表明 p40/ARPC1 在调节 Arp2/3 复合物介导的肌动蛋白组装中至少执行三个不同的基本功能：1）Arp2/3 复合物抑制自发成核，这需要与 p15/ARPC5 适当接触； 2) 通过与p19/ARPC2接触传播WASp激活信号； 3)通过延伸臂与WASp的VCA结构域的相互作用直接促进肌动蛋白成核。
The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by WASp (Wiskott Aldrich syndrome protein). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of the Arp2/3 complex have yet to be resolved. In particular, some of the individual subunits in the complex have not been assigned clear functional roles, including p40/ARPC1. Here, we dissected the structure and function of Saccharomyces cerevisiae p40/ARPC1, which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on p40/ARPC1 required for function in vivo: one site contacts p19/ARPC4, one contacts p15/ARPC5, and one site resides in an extended structural "arm" of p40/ARPC1. Using a novel strategy, we purified the corresponding lethal mutant Arp2/3 complexes from yeast and compared their actin nucleation activities. Lethal mutations at the contact with p19/ARPC4 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with p15/ARPC5 led to unregulated ("leaky") nucleation in the absence of WASp. Lethal mutations in the extended arm drastically reduced nucleation, and the same mutations disrupted the ability of the purified p40/ARPC1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/ARPC1 performs at least three distinct, essential functions in regulating Arp2/3 complex-mediated actin assembly: 1) suppression of spontaneous nucleation by the Arp2/3 complex, which requires proper contacts with p15/ARPC5; 2) propagation of WASp activation signals via contacts with p19/ARPC2; and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.