Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.
Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.
复制标题
DOI：
10.1083/jcb.131.4.1039
复制
发表时间：
1995-11
期刊：
影响因子：
--
通讯作者：
Hirokawa N
中科院分区：
文献类型：
--
作者：
Nakata T;Hirokawa N
在运动蛋白的研究中，机械化学偶联的分子机制及其在细胞中的作用是一个重要的问题。为了评估这些问题，我们将野生型和定点突变型激酶蛋白重链cDNA导入成纤维细胞，并分析了重组蛋白的行为和参与细胞器运输的机制。过表达野生型激酶显著促进细胞过程的延长。野生型激酶积聚在长过程的尖端，而激酶突变体在atp结合基序中包含T93N-或T93I突变，与细胞中心的微管紧密结合。这些突变的运动蛋白在体外可以与微管结合，但即使在ATP存在的情况下也不能与微管分离，并且在体外不支持微管运动，因此表明是严格型突变。从高尔基体到内质网的逆行运输，以及溶酶体的分散，被证明是一种微管依赖的，正端定向的运动。后者在严密性突变细胞中被选择性阻断，尽管溶酶体的微管负端定向运动不受影响。我们在体外实验中发现了使运动蛋白与微管处于强结合状态的点突变，并表明该突变在体内产生选择性阻断顺行溶酶体膜转运的显性效应。
In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.