Paul Aditya S, Pollard Thomas D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103, USA.
J Biol Chem. 2009 May 1;284(18):12533-40. doi: 10.1074/jbc.M808587200. Epub 2009 Feb 26.
Formin-homology (FH) 2 domains from formin proteins associate processively with the barbed ends of actin filaments through many rounds of actin subunit addition before dissociating completely. Interaction of the actin monomer-binding protein profilin with the FH1 domain speeds processive barbed end elongation by FH2 domains. In this study, we examined the energetic requirements for fast processive elongation. In contrast to previous proposals, direct microscopic observations of single molecules of the formin Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed that profilin is not required for formin-mediated processive elongation of growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin release the gamma-phosphate of ATP on average >2.5 min after becoming incorporated into filaments. Therefore, the release of gamma-phosphate from actin does not drive processive elongation. We compared experimentally observed rates of processive elongation by a number of different FH2 domains to kinetic computer simulations and found that actin subunit addition alone likely provides the energy for fast processive elongation of filaments mediated by FH1FH2-formin and profilin. We also studied the role of FH2 structure in processive elongation. We found that the flexible linker joining the two halves of the FH2 dimer has a strong influence on dissociation of formins from barbed ends but only a weak effect on elongation rates. Because formins are most vulnerable to dissociation during translocation along the growing barbed end, we propose that the flexible linker influences the lifetime of this translocative state.
来自formin蛋白的formin同源（FH）2结构域通过多次添加肌动蛋白亚基，在完全解离之前与肌动蛋白丝的带刺末端持续结合。肌动蛋白单体结合蛋白profilin与FH1结构域的相互作用加速了FH2结构域对带刺末端的持续延伸。在本研究中，我们研究了快速持续延伸的能量需求。与之前的观点相反，对用量子点标记的酿酒酵母formin Bni1p单分子进行的直接显微镜观察表明，profilin对于formin介导的带刺末端持续延伸不是必需的。由Bni1p和profilin聚合的ATP-肌动蛋白亚基在掺入丝状体后平均>2.5分钟释放ATP的γ-磷酸。因此，肌动蛋白释放γ-磷酸并不驱动持续延伸。我们将实验观察到的多个不同FH2结构域的持续延伸速率与动力学计算机模拟进行了比较，发现仅肌动蛋白亚基的添加可能为FH1FH2-formin和profilin介导的丝状体快速持续延伸提供能量。我们还研究了FH2结构在持续延伸中的作用。我们发现连接FH2二聚体两半的柔性接头对formin从带刺末端的解离有很大影响，但对延伸速率的影响较弱。由于formin在沿生长的带刺末端转运过程中最容易解离，我们提出柔性接头影响这种转运状态的寿命。
