Distinct Functions of Nucleotide-Binding/Hydrolysis Sites in the Four AAA Modules of Cytoplasmic Dynein

详细信息    查看全文

• 作者：Takahide Kon ; Masaya Nishiura ; Reiko Ohkura ; Yoko Y. Toyoshima ; and Kazuo Sutoh
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：September 7, 2004
• 年：2004
• 卷：43
• 期：35
• 页码：11266 - 11274
• 全文大小：310K
• 年卷期：v.43,no.35(September 7, 2004)
• ISSN：1520-4995

文摘

Cytoplasmic dynein is a microtubule-based motor protein that is responsible for mostintracellular retrograde transports along microtubule filaments. The motor domain of dynein contains sixtandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first fourcontaining predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiplenucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains inwhich mutations were introduced to block nucleotide binding at each of the four AAA modules, and thenexamined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state evenin the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity formicrotubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, butretained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-foldslower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparentbinding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubulesliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essentialfor the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysissites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but iscritical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.