The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.

During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8 Kif18A has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin Kif18A has the unexpected function of suppressing chromosome movements. Based on these findings, we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning.

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.