Quintero-Monzon Omar, Jonasson Erin M, Bertling Enni, Talarico Lou, Chaudhry Faisal, Sihvo Maarit, Lappalainen Pekka, Goode Bruce L
Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
J Biol Chem. 2009 Apr 17;284(16):10923-34. doi: 10.1074/jbc.M808760200. Epub 2009 Feb 6.
Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.
Srv2/环化酶相关蛋白几乎在所有植物、动物和真菌生物体中都有表达，在促进肌动蛋白解聚因子/丝切蛋白介导的肌动蛋白周转中发挥着保守作用。这是通过Srv2从ADP-肌动蛋白单体中回收丝切蛋白以及促进肌动蛋白单体上的核苷酸交换（将ATP交换为ADP）的能力来实现的。尽管在促进肌动蛋白周转方面具有这一重要且普遍的作用，但Srv2功能的潜在机制仍然难以捉摸。先前的研究已经证明Srv2的G-肌动蛋白结合C末端一半具有关键的功能作用。在这里，我们描述了Srv2的N末端一半在体内驱动肌动蛋白周转方面同样重要的作用。我们将这种活性定位到Srv2的N末端二聚体螺旋折叠结构域上一个保守的表面残基区域，并且我们表明这个功能位点与丝切蛋白-肌动蛋白复合物相互作用。此外，我们表明这个位点对于Srv2在体外加速丝切蛋白介导的肌动蛋白周转至关重要。在丝切蛋白的一个保守表面上鉴定出了一个同源的Srv2结合位点，这表明这种功能可能延伸到其他生物体。另外，我们的分析揭示Srv2的高阶寡聚化依赖于其N末端预测的卷曲螺旋结构域，并且寡聚化在体外和体内优化了Srv2的功能。基于这些数据，我们提出了一个关于Srv2促进肌动蛋白周转机制的修订模型，其中其N末端和C末端两半的协同活动催化了丝切蛋白和肌动蛋白单体循环利用中的连续步骤。
