Biochemical Characterization of the Rice Kinesin O12 and N14 Belonging to Kinesin-14 Family

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. Kinesins derived from vertebrate have been well studied on their characterization. However, not so many studies for kinesins of plants have been done yet. Recently, the genome sequences of rice were completed. Bioinformatics analyses revealed that at least 41 kinesin-related proteins were encoded on the rice genome. In this study, we focused on the two rice kinesins; O12 and N14 that belong to kinesin-14 family. Previously, we have expressed the novel rice kinesin O12 by E.coli. Biochemical studies of the O12 motor domain demonstrated that O12 has very unique properties, which may reflect the plant specific physiological role. We have also succeeded to express another rice specific kinesins N14. O12 is the C-terminal motor domain. On the other hand, the motor domains of N14 are found at the central region in the primary structure. The biochemical characterizations of the kinesin N14 motor were studied. ATPase activities and interaction with fluorescent ATP analogues, NBD-ATP were analyzed and compared with other rice kinesins and conventional mouse kinesin. Interestingly, microtubule-dependent steady state ATPase activity of N14 motor domain was extremely lower than that of other kinesins. Kinetic analyses using stopped-flow also demonstrated that ATP binding to N14 in the presence of microtubule was extremely slow in comparison with other kinesins. It is suggested that the rice kinesin N14 may have unique enzymatic properties that are obviously different from O12.