Sakato Miho, King Stephen M
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030-3305, USA.
J Biol Chem. 2003 Oct 31;278(44):43571-9. doi: 10.1074/jbc.M305894200. Epub 2003 Aug 14.
The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.
衣藻外动力蛋白臂包含三种不同的重链（α、β和γ），它们表现出不同的运动特性。LC4蛋白与γ重链相关联，它能以KCa = 3×10⁻⁵ m的亲和力结合1 - 2个Ca²⁺，有人提出它作为一种传感器，可根据鞭毛内Ca²⁺水平的变化来调节动力蛋白的运动功能。在这里，我们通过遗传学方法剖析外臂，以产生包含不同运动单元组合的亚颗粒，并在与微管蛋白和ATP预孵育之前和之后评估这些复合物的微管结合特性，ATP用于抑制对ATP不敏感的（结构）微管结合。我们观察到，α重链在体外表现出占主导地位的不依赖Ca²⁺的ATP敏感的微管结合活性，这种活性会被微管蛋白附着到结构微管结合域所抑制。此外，我们表明，仅包含β和γ重链的动力蛋白亚颗粒的ATP敏感微管结合，在Ca²⁺浓度低于pCa 6时不会发生，但在pCa 5以上时被最大程度激活。在β重链微管结合区域有小缺失的突变动力蛋白或既缺乏α重链又缺乏β重链运动结构域的动力蛋白中未观察到这种活性。这些发现有力地表明，Ca²⁺直接与动力蛋白复合物的一个组分结合，调节β重链与微管之间的ATP敏感相互作用，并由此得出一个关于外动力蛋白臂内单个运动单元如何被控制的模型。
