Nozomi Umezu

Characterization of a novel rice kinesin O12 with a calponin homology domain

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.

Characterization of the Plant-Specific Rice Kinesins

Kinesin is a motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics and signal transduction. Kinesin converts chemical energy from ATP into mechanical force. Kinesin family is classified into some subfamilies. Some species of kinesin derived from vertebrate have been well studied. However, not so many studies for kinesins of plants have been done yet. Recently, the genome sequences of rice were completed. Bioinformatical 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; 1. O12 that has a calponin homology domain, 2. K23 that belongs to At1 subfamily in kinesin-7. The cDNAs of the kinesin motor domain was subcloned into expression vector pET and transformed into E. coli BL21 (DE3). kinesin motor domains were expressed and purified by Co-NTA column. The biochemical characterizations of the two rice kinesins were studied. The microtubule-dependent ATPase activity of the two rice kinesins motor domains were 30∼60-fold lower than that of conventional kinesin. Kinetic analyses using stopped-flow demonstrated that ATP binding to O12 in the absence of microtubule was extremely slow compared with that of conventional kinesin. While, ATP binding to K23 was not accelerated by microtubule. Furthermore, interestingly ATPase activity of O12 in the absence microtubule regulated by actin. The O12-tail fused with GFP was observed to localize in the actin filament in the onion cell. The two plant specific rice kinesin O12 and K23 were shown to have unique enzymatic properties.