Studies on Molecular Mechanism of Force Generation by Recombinant Dynein

• Project/Area Number: 15370063
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: The University of Tokyo
• Principal Investigator: SUTOH Kazuo The University of Tokyo, Graduate School of Arts and Science, Professor, 大学院・総合文化研究科, 教授 (20111453)
• Co-Investigator(Kenkyū-buntansha): KON Takahide The University of Tokyo, Graduate School of Arts and Science, Research Associate, 大学院・総合文化研究科, 助手 (30332620)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥15,000,000 (Direct Cost: ¥15,000,000); Fiscal Year 2004: ¥7,300,000 (Direct Cost: ¥7,300,000); Fiscal Year 2003: ¥7,700,000 (Direct Cost: ¥7,700,000)
• Keywords: motor proteins / intracellular transport / sliding motion / power stroke / dynein / myosin / kinesin / 分子モーター / 組み換え体 / 微小管 / 遺伝子組換え / AAAファミリータンパク質

Research Abstract

We have succeeded to express recombinant fragment of Dictyostelium cytoplasmic dynein in Dictyostelium cells (Nishiura et al. J.Biol.Chem (2004)). The expressed dynein fragment with molecular mass of 380kDa is consisted of the C-terminal heavy chain fragment, and maintains full motor activities : the fragment, a single-headed dynein motor, has high microtubule-activated ATPase activity (120 s^<-1>), and drives microtubule sliding at 3 μm/sec. By microtubule-landing assays, the sliding is shown to be non-processive, and the duty ratio is determined as 0.37. By combining these values, the step size is expected to be 8 nm. Thus, this active recombinant cytoplasmic dynein fragment behaves like the single-headed kinesin, another microtubule-based motor. By using this unique expression system, we have examined functional roles of four potential ATPase sites in the AAA+-ring of the dynein motor domain. We introduced point mutations in the highly conserved Walker A and Walker B sequences in these four potential ATPase sites, and examined their basal and microtubule-activated ATPase activities as well as their motility. We have found that the first AAA+ module has the primary ATPase site responsible for the force generation, and the third AAA+ module has the ATPase site tightly coupled with the former (Kon et al., Biochemistry (2004)). We have then developed the GFP-based FRET system to detect ATP hydrolysis cycle-dependent structural transitions of dynein, which may correspond to power stroke (Kon et al., Nature Structural and Molecular Biology (2005)). We identified two distinct structural states that have distinct fluorescence spectra due to FRET between GFP and BFP attached to two locations on the active dynein motor. We also correlated these two states to intermediate states of cyclic ATP hydrolysis at the dynein primary ATPase site.

Research Products

• [Journal Article] ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein (2005)
  • Author(s): Kon, T., Mogami, T., Ohkura, R., Nishiura, M., Sutoh, K.
  • Journal Title: Nature Structural and Molecular Biology (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein. (2005)
  • Author(s): Takahide Kon, Toshifumi Mogami, Reiko Ohkura, Masaya Nishiura, Kazuo Sutoh
  • Journal Title: Nature Structural and Molecular Biology (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The N-Terminal Domain of MYO18A Has an ATP-Insensitive Actin-Binding Site. (2005)
  • Author(s): Yasushi Isogawa, Takahide Kon, Takeshi Inoue, Reiko Ohkura, Hisashi Yamakawa, Osamu Ohara, Kazuo Sutoh
  • Journal Title: Biochemistry 44 Pages: 6190-6196
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The N-Terminal Domain of MYO18A Has an ATP-Insensitive Actin-Binding Site (2004)
  • Author(s): Isogawa, Y., Kon, T., Inoue, T., Ohkura, R., Yamakawa, H., Ohara, O., Sutoh, K.
  • Journal Title: Biochemistry 44 Pages: 6190-6196
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein, as revealed by biochemical characterizations of recombinant fragments. (2004)
  • Author(s): Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: Biochemistry 43 Pages: 11266-11274
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules. (2004)
  • Author(s): Nishiura, M., Kon, T., Shiroguchi, K., Ohkura, R., Shima, T., Toyoshima, Y.Y., Sutoh.K.
  • Journal Title: J.Biol.Chem. 279 Pages: 22799-22802
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A Novel Actin-bundling Kinesin-related Protein from Dictyostelium discoideum (2004)
  • Author(s): Iwao, S., Ishiji, A., Mabuchi, I., Sutoh, K.
  • Journal Title: J.Biol.Chem. 279 Pages: 4696-4704
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Distinct Functions of Nucleotide-Binding/Hydrolysis Sites in the Four AAA Modules of Cytoplasmic Dynein. (2004)
  • Author(s): Takahide Kon, Masaya Nishiura, Reiko Ohkura, Yoko Y.Toyoshima, Kazuo Sutoh
  • Journal Title: Biochemistry 43 Pages: 11266-11274
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A Single-headed Recombinant Fragment of Dictyostelium Cytoplasmic Dynein Can Drive the Robust Sliding of Microtubules. (2004)
  • Author(s): Masaya Nishiura, Takahide Kon, Katsuyuki Shiroguchi, Reiko Ohkura, Tomohiro Shima, Yoko Y.Toyoshima, Kazuo Sutoh
  • Journal Title: The Journal of Biological Chemistry Vol.279, No.22 Pages: 22799-22802
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A Novel Actin-bundling Kinesin-related Protein from Dictyostelium discoideum. (2004)
  • Author(s): Sosuke Iwai, Atsushi Ishiji, Issei Mabuchi, Kazuo Sutoh
  • Journal Title: The Journal of Biological Chemistry Vol.279, No.6 Pages: 4696-4704
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein, as revealed by biochemical charscterizations of recombinant fragments. (2004)
  • Author(s): Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: Biochemistry 43 Pages: 11266-11274
• [Journal Article] A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules. (2004)
  • Author(s): Nishiura, M., Kon, T., Shiroguchi, K., Ohkura, R., Shima, T., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: J Biol.Chem. 279 Pages: 22799-22802
• [Journal Article] A Novel Actin-bundling Kinesin-related Protein from Dictyostelium ciscoideum (2004)
  • Author(s): Iwai, S., Ishiji, A., Mabuchi, I., Sutoh, K.
  • Journal Title: J Biol.Chem. 279 Pages: 4696-4704
• [Journal Article] Dictyostelium Myosin II Mutations That Uncouple the Converter Swing and ATP Hydrolysis Cycle (2003)
  • Author(s): Sasaki, N., Ohkura, R., Sutoh, K.
  • Journal Title: Biochemistry 42 Pages: 90-95
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dictyostelium Myosin II Mutations That Uncouple the Converter Swing and ATP Hydrolysis Cycle. (2003)
  • Author(s): Naoya Sasaki, Reiko Ohkura, Kazuo Sutoh
  • Journal Title: Biochemistry 42 Pages: 90-95
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sousuke Iwai, Atsushi Ishiji, Issei Mabuchi, Kazuo Sutoh: "A novel actin-binding kinesin-related protein from Dictyostelium discoideum"J.Biol.Chem.. 279. 4696-4704 (2004)
• [Publications] Sasaki, N., Ohkura, R., Sutoh, K.: "Dictyostelium Myosin II Mutations That Uncouple the Converter Swing and ATP Hydrolysis Cycle"Biochemistry. 42. 90-95 (2003)