The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein.
The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this "power stroke," dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steady-state and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a K(d) of approximately 0.2 microM, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a K(d) of >10 microM. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.
Imamula
Kenji
K
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153-8902, Japan.
Kon
Takahide
T
Ohkura
Reiko
R
Sutoh
Kazuo
K
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D003593:Cytoplasm / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000227:Adenine Nucleotides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D004398:Dyneins