Takahashi Yoshinori, Edamatsu Masaki, Toyoshima Yoko Y
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12865-9. doi: 10.1073/pnas.0403429101. Epub 2004 Aug 23.
Cytoplasmic dynein is a minus-end-directed microtubule motor involved in numerous essential processes within eukaryotic cells, such as nuclear segregation and trafficking of intracellular particles. The motor domain of the dynein heavy chain comprises six tandemly linked AAA (ATPase associated with diverse cellular activities) modules (AAA1-AAA6). The first four modules include nucleotide-binding sites (Walker A or P-loop motifs), and each of the four sites appears to bind ATP. However, the role and the function of each binding site are unknown. Especially, the question of which P-loops are ATP-hydrolyzing sites has not been answered, because it is difficult to measure the ATPase activity of each P-loop. Here, we purified several truncated Saccharomyces cerevisiae cytoplasmic dynein fragments and their mutants expressed in Escherichia coli and then measured their ATPase activities. Our results suggest that there are multiple ATP-binding sites that have abilities to hydrolyze ATP in cytoplasmic dynein. Furthermore, a single AAA module is insufficient for ATP hydrolysis, and the adjacent module facing the ATP-binding site is necessary for ATP-hydrolyzing activity.
胞质动力蛋白是一种向微管负端移动的分子马达，参与真核细胞内众多重要过程，如核分离和细胞内颗粒运输。动力蛋白重链的马达结构域由六个串联的AAA（与多种细胞活动相关的ATP酶）模块（AAA1 - AAA6）组成。前四个模块包含核苷酸结合位点（沃克A或P环基序），四个位点中的每一个似乎都能结合ATP。然而，每个结合位点的作用和功能尚不清楚。特别是，哪个P环是ATP水解位点的问题尚未得到解答，因为很难测量每个P环的ATP酶活性。在这里，我们纯化了几种在大肠杆菌中表达的截短的酿酒酵母胞质动力蛋白片段及其突变体，然后测量了它们的ATP酶活性。我们的结果表明，胞质动力蛋白中有多个具有ATP水解能力的ATP结合位点。此外，单个AAA模块不足以进行ATP水解，面向ATP结合位点的相邻模块对于ATP水解活性是必需的。
