Chaudhry Faisal, Guérin Christophe, von Witsch Matthias, Blanchoin Laurent, Staiger Christopher J
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2064, USA.
Mol Biol Cell. 2007 Aug;18(8):3002-14. doi: 10.1091/mbc.e06-11-1041. Epub 2007 May 30.
The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP-actin.
肌动蛋白细胞骨架驱动细胞器运动，协调对非生物胁迫的反应，并产生一系列令人惊叹的细胞形状。支撑肌动蛋白细胞骨架这些多样功能的是几十种辅助蛋白，它们协调肌动蛋白丝的动态变化并构建高阶组装体。许多来自植物界的肌动蛋白结合蛋白已得到表征，其功能往往与哺乳动物和真菌中的对应蛋白惊人地不同。腺苷酸环化酶相关蛋白（CAP）最近已被证明是体内和体外肌动蛋白动态变化的重要调节因子。cap突变体植物中肌动蛋白组织的破坏表明肌动蛋白动态变化存在缺陷，或者肌动蛋白亚基组装和解聚成丝的过程受到调控。目前的肌动蛋白动态变化模型认为，肌动蛋白解聚因子（ADF）/丝切蛋白从丝末端去除ADP-肌动蛋白亚基，而脯氨酸丰富蛋白通过增强核苷酸交换并将亚基递送到丝的带刺末端，用ATP为这些单体重新充电。然而，植物脯氨酸丰富蛋白缺乏刺激肌动蛋白上核苷酸交换的基本能力，这表明植物中可能还存在一个尚未被发现的缺失环节。在这里，我们表明拟南芥CAP1（AtCAP1）是一种丰富的细胞质蛋白；在悬浮细胞中，它与总肌动蛋白的比例为1:3M。AtCAP1对ADP-和ATP-单体肌动蛋白具有同等亲和力（解离常数约为1.3 microM）。AtCAP1与ATP-肌动蛋白单体的结合会抑制聚合，这与AtCAP1是一种肌动蛋白隔离蛋白一致。然而，我们证明AtCAP1是第一种能提高肌动蛋白上核苷酸交换速率的植物蛋白。即使在存在ADF/丝切蛋白的情况下，AtCAP1也能为肌动蛋白单体重新充电，并可能为脯氨酸丰富蛋白提供一个可聚合的亚基库，以便添加到丝末端。在周转试验中，植物脯氨酸丰富蛋白、ADF和CAP协同作用，促进亚基通过肌动蛋白丝带刺末端的通量。总的来说，这些结果以及我们对其他肌动蛋白结合蛋白的了解表明，CAP1是调节未聚合ATP-肌动蛋白库的核心参与者。
