Belmont L D, Drubin D G
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
J Cell Biol. 1998 Sep 7;142(5):1289-99. doi: 10.1083/jcb.142.5.1289.
Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.
带有缬氨酸159突变为天冬酰胺（V159N）的肌动蛋白形成的肌动蛋白丝解聚缓慢，原因是在无机磷酸释放后未能发生构象变化。在此，我们证明这种肌动蛋白的表达会导致体内肌动蛋白动力学降低，并且我们利用这一特性来研究快速肌动蛋白丝周转的作用。表达V159N突变体（act1 - 159）作为其唯一肌动蛋白来源的酵母菌株，与野生型酵母相比，具有更大的皮质肌动蛋白斑和更多的肌动蛋白电缆。快速肌动蛋白动力学对于皮质肌动蛋白斑的运动性或细胞极性的建立并非必不可少。然而，act1 - 159菌株中的液相内吞作用存在缺陷。act1 - 159与肌动蛋白解聚因子和肌动蛋白单体结合蛋白突变体具有合成致死性，支持了所有这些基因突变都会损害聚合/解聚循环的结论。相比之下，act1 - 159部分抑制了原肌球蛋白突变体的温度敏感性，以及在肌动蛋白结合蛋白、Mdm20p和原肌球蛋白缺失突变体中所见的细胞质电缆的丧失，这表明这些肌动蛋白结合蛋白具有细丝稳定功能。对这些双突变细胞中电缆的分析支持了肌动蛋白结合蛋白在组织细胞质电缆中的作用，以及Mdm20p和原肌球蛋白在将肌动蛋白解聚因子排除在电缆之外的作用。
