Mackey Andrew T, Gilbert Susan P
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
J Biol Chem. 2003 Feb 7;278(6):3527-35. doi: 10.1074/jbc.M206219200. Epub 2002 Nov 24.
Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with Ncd showed that both motor domains were required for ATP-dependent motor domain detachment from the microtubule. We have initiated a series of experiments to compare the mechanistic requirements for Kar3 motility in direct comparison to Ncd. The results presented here show that the single motor domain of Kar3 (Met(383)-Lys(729)) exhibits characteristics similar to monomeric Ncd. The microtubule-activated steady-state ATPase cycle of Kar3 (k(cat) = 0.5 s(-1)) is limited by ADP release (0.4 s(-1)). Like monomeric Ncd, Kar3 does not readily detach from the microtubule with the addition of MgATP. These results show that the single motor domain of Kar3 is not sufficient for ATP-dependent microtubule dissociation, suggesting that structural elements outside of the catalytic core are required for the cyclic interactions with the microtubule for force generation.
Kar3是一种参与酿酒酵母减数分裂和有丝分裂的负端定向微管马达蛋白。与另一种特征明确的负端定向马达蛋白果蝇Ncd（它是一种同二聚体）不同，Kar3是一种异二聚体，具有单个马达结构域以及相关多肽Cik1或Vik1。我们对Ncd进行的机制研究表明，两个马达结构域对于ATP依赖的马达结构域从微管上脱离都是必需的。我们已经启动了一系列实验，将Kar3的运动机制要求与Ncd进行直接比较。此处呈现的结果表明，Kar3的单个马达结构域（Met(383)-Lys(729)）表现出与单体Ncd相似的特征。Kar3的微管激活稳态ATP酶循环（k(cat)=0.5 s(-1)）受ADP释放（0.4 s(-1)）限制。与单体Ncd一样，添加MgATP后Kar3不会轻易从微管上脱离。这些结果表明，Kar3的单个马达结构域不足以实现ATP依赖的微管解离，这表明催化核心之外的结构元件对于与微管进行循环相互作用以产生力是必需的。
