Kinetic Characterization of Novel Rice Plant Kinesin E11

Abstract

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures. In this study, we characterized biochemical and kinetic properties of another rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E.coli expression system and purified with Co-chelate column. The fluorescent ATP analogue, Mant-ATP was employed for the kinetic study. We observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters were analyzed by monitoring the FRET using stopped flow apparatus. The binding rate and dissociation rate were measured, and compared with those of other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are faster than those of other rice plant specific kinesin. We also examined the motility activity of E11 with in vitro microtubule gliding assay. The motility assay revealed that E11 has relatively higher motor activity than other rice kinesins.