A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules.
A cytoplasmic dynein is a microtubule-based motor protein involved in diverse cellular functions, such as organelle transport and chromosome segregation. The dynein has two ring-shaped heads that contain six repeats of the AAA domain responsible for ATP hydrolysis. It has been proposed that the ATPase-dependent swing of a stalk and a stem emerging from each of the heads generates the power stroke (Burgess, S.A. (2003) Nature 421, 715-718). To understand the molecular mechanism of the dynein power stroke, it is essential to establish an easy and reproducible method to express and purify the recombinant dynein with full motor activities. Here we report the expression and purification of the C-terminal 380-kDa fragment of the Dictyostelium cytoplasmic dynein heavy-chain fused with an affinity tag and green fluorescent protein. The purified single-headed recombinant protein drove the robust minus-end-directed sliding of microtubules at a velocity of 1.2 microm/s. This recombinant protein had a high basal ATPase activity (approximately 4s(-1)), which was further activated by >15-fold on the addition of 40 microM microtubules. These results show that the 380-kDa recombinant fragment retains all the structures required for motor functions, i.e. the ATPase activity highly stimulated by microtubules and the robust motility.
Nishiura
Masaya
M
Department of Life Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
Kon
Takahide
T
Shiroguchi
Katsuyuki
K
Ohkura
Reiko
R
Shima
Tomohiro
T
Toyoshima
Yoko Y
YY
Sutoh
Kazuo
K
D000818:Animals; D004023:Dictyostelium; D004398:Dyneins / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000502:physiology*; D010446:Peptide Fragments / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004398:Dyneins