Multiple ATP-hydrolyzing sites that potentially function in cytoplasmic dynein
- Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
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Edited by Ronald D. Vale, University of California, San Francisco, CA, and approved July 25, 2004 (received for review May 14, 2004)
Abstract
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.
Footnotes
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↵ * To whom correspondence should be addressed. E-mail: cyytoyo{at}mail.ecc.u-tokyo.ac.jp.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: AAA, ATPases associated with diverse cellular activities; BCCP, biotin carboxyl carrier protein.
- Copyright © 2004, The National Academy of Sciences
