Karki S, Holzbaur E L
Cell Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, USA.
J Biol Chem. 1995 Dec 1;270(48):28806-11. doi: 10.1074/jbc.270.48.28806.
We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150Glued, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150Glued affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150Glued affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150Glued may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150Glued component of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150Glued, which is predicted to form an alpha-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport.
我们使用亲和层析法来探究胞质动力蛋白与动力蛋白激活蛋白之间的直接结合相互作用。结果发现，纯化的胞质动力蛋白能与动力蛋白激活蛋白复合物中最大的多肽p150Glued的亲和柱结合。为了测试这种相互作用的特异性，我们将大鼠脑胞质溶胶加载到p150Glued亲和柱上，观察到胞质溶胶中的胞质动力蛋白被特异性地保留在柱上。用过量的外源性动力蛋白中间链对p150Glued亲和基质进行预孵育，导致动力蛋白结合显著减少，这表明p150Glued可能通过该多肽与动力蛋白相互作用。因此，我们构建了重组动力蛋白中间链的亲和柱，并观察到动力蛋白激活蛋白从大鼠脑胞质溶胶中被保留下来。这些结果表明，天然的动力蛋白和动力蛋白激活蛋白复合物能够在体外通过动力蛋白中间链与动力蛋白激活蛋白复合物的p150Glued组分的直接结合介导直接相互作用。我们已经将这种相互作用的位点定位到p150Glued的氨基末端区域，该区域预计会形成一个α螺旋卷曲螺旋结构。动力蛋白 - 动力蛋白激活蛋白相互作用的调节可能是胞质动力蛋白介导的囊泡运输控制机制的关键。
