Analysis of Calcium/Calmodulin Regulation of a Plant Kinesin Using Co-Sedimentation and ATPase Assays

Abstract

Kinesins, a superfamily of microtubule motor proteins, are implicated in regulating a number of fundamental cellular and developmental processes including intracellular transport of vesicles and organelles, mitotic and meiotic spindle formation and elongation, chromosome segregation, germplasm aggregation, microtubule (MT) organization and dynamics, and intraflagellar transport. Analysis of all the completed genomes of eukaryotes has revealed that Arabidopsis, a flowering plant, has more kinesins than any other organism. Although a complete inventory of kinesins in a number of organisms has been reported, the function and regulation of kinesins in general and plant kinesins in particular are poorly understood. In our screen of an expression library with a labeled calmodulin, we isolated a novel plant kinesin (kinesin-like calmodulin-binding protein, KCBP) from plants, which interacts with calmodulin in a calcium-dependent manner. This chapter describes the methods used in elucidating the regulation of this motor protein by calcium/calmodulin.