MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1.08 ؎ 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force < < 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.Conventional kinesin is a protein machine that steps along the surface of a microtubule as it carries a membrane-bound organelle toward the periphery of a cell (1-3). The size of the steps is ϳ8 nm (4,5). This is the distance between consecutive binding sites along the microtubule protofilament (6, 7), and a single kinesin molecule can take hundreds of steps without detaching (8, 9), even against opposing loads as high as ϳ6 pN (10 -12). The steps are driven by the hydrolysis of ATP; kinesin is an ATPase (13) whose speed of movement increases linearly with ATP concentration until it approaches a maximum of about 800 nm/s (8, 14), and AMP-PNP, 1 a nonhydrolyzable analog of ATP, arrests movement (15, 16). Despite intensive biochemical, biophysical, and structural investigations over the last few years, there remains considerable uncertainty as to the mechanism by which the stepping is coupled to ATP hydrolysis.In this work, we address the question: what is the stoichiometry of kinesin? In other words, how many steps does kinesin take for each ATP that it hydrolyzes? This question is important because it tests different models proposed to explain how motor proteins such as kinesin and myosin work. For example, one class of "thermal ratchet" models predicts that the stoichiometry is Յ0.5 steps/ATP (17, 18); these models postulate that ATP hydrolysis rectifies a diffusive motion in such a way that a step only occurs if the diffusion is toward the next binding site. Because there is an equal probability that the motor diffuses away from the next binding site (i.e. in the wrong direction), this model predicts that on average at least two molecules of ATP are hydrolyzed per forward step. A stoichiometry of less than one could also be due to "futile" hydrolysis cycles, those that fail to produce steps and lead to "slippage" between the mechanical and chemical cycles. Other models postulate a stoichiometry greater than 1 and have been...

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.

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