Mechanism of formin-induced nucleation of actin filaments
Mechanism of formin-induced nucleation of actin filaments
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DOI：
10.1021/bi026520j
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发表时间：
2003-01-21
期刊：
影响因子：
2.9
通讯作者：
Zigmond, SH
中科院分区：
文献类型：
--
作者：
Pring, M;Evangelista, M;Zigmond, SH
含有FH I FH 2结构域的酵母GABm I的片段增加来自纯G-肌动蛋白的丝成核速率[Pruyne等人（2002）Science 297，612-615]。为了确定成核机制，我们将Bni I FH I FH 2诱导的聚合的G-肌动蛋白依赖性与理论模型进行了比较。数据最佳地拟合表明Bni I FH I FH 2稳定肌动蛋白二聚体的模型。我们还表明，成核增加的平方根的Bni 1FH 1FH 2浓度。我们证明，这种关系是预期的任何这样的成核剂，独立的核大小。富含脯氨酸的FH 1结构域结合profilin，并且该结构域的缺失降低了profilin-actin对成核的贡献。支持的作用，为profilin结合到FH I结构域中的细丝成核的Bni 1FH 1FH 2不能利用突变体profilin，H133 S profilin，与缺陷的结合到聚脯氨酸。BNi I FH I FH 2部分抑制倒刺末端伸长，并且我们发现聚合和解聚的速率常数均降低约50%。Bni 1FH 1FH 2对尖端动力学或临界浓度没有影响。为了研究这些活性所需的Bni I的结构域，实验全部用单独的FH 2结构域重复。FH 2结构域与FH 1 FH 2结构域一起在抑制倒钩末端动力学方面一样有效;它作为成核剂的有效性较低，但该机制再次最适合二聚体稳定化。
A fragment of the yeast formin Bni I containing the FH I FH2 domains increases the rate of filament nucleation from pure G-actin [Pruyne et al. (2002) Science 297, 612-615]. To determine the mechanism of nucleation, we compared the G-actin dependence of Bni I FH I FH2-induced polymerization with theoretical models. The data best fit a model suggesting, that Bni I FH I FH2 stabilizes an actin dimer. We also show that nucleation increases with the square root of the Bni1FH1FH2 concentration. We demonstrate that this relationship is expected for any such nucleator, independent of nucleus size. The proline-rich FH1 domain binds profilin, and deletion of this domain decreases the contribution of profilin-actin to the nucleation. A role for profilin binding to the FH I domain in filament nucleation was supported by the inability of Bni1FH1FH2 to utilize a mutant profilin, H133S profilin, with defective binding to polyproline. Bni I FH I FH2 partially inhibits barbed-end elongation, and we find that the rate constants for both polymerization and depolymerization are decreased by approximately 50%. Bni1FH1FH2 has no effect on pointed-end kinetics or on the critical concentration. To investigate the domains of Bni I required for these activities, the experiments were all duplicated with the FH2 domain alone. The FH2 domain is as effective as the FH I FH2 domains together in inhibiting barbed-end kinetics; it is less effective as a nucleator but the mechanism is again best fit by dimer stabilization.