Abstract
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.
Highlights
The 380-kDa fragment of the Dictyostelium dynein heavychain has been successfully expressed in Dictyostelium cells [13]
Electron microscopic studies on the fragment have shown that it has a single ring-shaped head and a stalk protruding from it [14]. This fragment binds to microtubules in an ATPsensitive fashion and is susceptible to VO4-mediated photocleavage [13], an indication that the fragment may have ATPase activity. It remains to be shown whether this recombinant dynein fragment is an active motor with microtubule-activated ATPase activity and motility
We have the 380-kDa recombinant fragment retains all the struc- established an expression and purification system of the singletures required for motor functions, i.e. the ATPase headed Dictyostelium 380-kDa dynein fragment fused with the activity highly stimulated by microtubules and the N-terminal His tag and GFP1 and have robust motility
Summary
The 380-kDa fragment of the Dictyostelium dynein heavychain has been successfully expressed in Dictyostelium cells [13]. 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 m/s.
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