Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA 02454, USA.
Mol Biol Cell. 2011 Nov;22(21):4016-28. doi: 10.1091/mbc.E11-05-0404. Epub 2011 Aug 31.
Formins are a conserved family of actin assembly-promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.
formin 是一类肌动蛋白组装促进因子，具有多种生物学功能，但它们在体内的活性如何调节还不是很清楚。在酿酒酵母中，formin Bni1 和 Bnr1 对于肌动蛋白电缆的组装和极化细胞生长是必需的。适当的电缆组装还需要 Bud6。先前的研究表明，Bud6 增强了体外 Bni1 介导的肌动蛋白组装，但这种活性的生化机制和体内作用尚不清楚。在这里，我们证明 Bud6 特异性地刺激 Bni1 介导的肌动蛋白组装的成核阶段，而不是延伸阶段，将 Bud6 定义为成核促进因子（NPF），并将其作用与 Profilin 区分开来。我们生成了 Bud6 的等位基因，使 Bud6 与 Bni1 和 G-actin 的相互作用解耦，发现这两种相互作用对 NPF 活性都是至关重要的。我们的数据表明，Bud6 通过将肌动蛋白单体募集到 Bni1 上来促进丝状核的形成。对相同等位基因的遗传分析表明，Bud6 对形成素活性的调节对于体内正常水平的肌动蛋白电缆组装是至关重要的。我们的研究结果表明，Bud6 与 WASP 之间以及 Bud6 与 Spire 等与formin 相互作用的其他 NPF 之间存在重要的机制相似性。
