DNA AND CELL BIOLOGY Volume 19, Number 9, 2000 Mary Ann Liebert, Inc. Pp. 567–578 A Novel Calcium/Calmodulin-Regulated Kinesin-Like Protein is Highly Conserved between Monocots and Dicots SALAH E. ABDEL-GHANY and A.S.N. REDDY ABSTRACT Recently, a novel kinesin-like protein (KCBP) that is regulated by Ca 21 /calmodulin was isolated from dicot plants. A homolog of KCBP has not been reported in monocots. To determine if this motor protein is present in phylogenetically divergent flowering plants, Arabidopsis KCBP cDNA was used as a probe to screen a ge- nomic library of maize, an evolutionarily divergent species. This screening resulted in isolation of a KCBP homolog. Comparison of the predicted amino acid sequence of the KCBP from maize (ZmKCBP), a mono- cot, with the previously reported KCBP sequences from dicot species showed that the amino acid sequence, domain organization, and gene structure are highly conserved between monocots and dicots. The C-terminal region of ZmKCBP, containing the motor domain and the calmodulin-binding domain, and the N-terminal tail, with a myosin tail homology region (MyTH4) and talin-like region, showed strong sequence similarity to the KCBP homolog from dicots. However, the coiled-coil region is less conserved between monocots and di- cots. The ZmKCBP gene contained 22 exons and 21 introns. The location of 19 of the 21 introns of ZmKCBP is also conserved. The ZmKCBP protein is encoded by a single gene and expressed in all tissues. Affinity-pu- rified antibody to the calmodulin-binding domain of Arabidopsis KCBP detected a protein in both the solu- ble and the microsomal fractions. The C-terminal region of ZmKCBP, containing the motor and calmodulin- binding domains, bound calmodulin in the presence of calcium and failed to bind in the presence of EGTA. The ZmKCBP, along with other KCBPs from dicots, was grouped into a distinct group in the C-terminal sub- family of kinesin-like proteins. These data suggest that the KCBP is ubiquitous and highly conserved in all flowering plants and the origin of KCBP predated the divergence of monocots and dicots. 567 INTRODUCTION M OLECULAR MOTORS ARE INVOLVED in a wide range of fun- damental cellular processes, including intracellular or- ganelle movement, cell division, motility, and muscle contrac- tion (Langford, 1995; Hirokawa, 1998). These motor proteins contain a specialized enzymatic domain called the motor do- main that hydrolyzes ATP and uses the derived chemical en- ergy to move along a cytoskeletal polymer (actin in the case of myosins,microtubulesin the case of kinesinand dynein)(Hack- ney, 1996). The myosin motors move toward the plus end of actin filaments (Brown, 1999). The microtubule motors, on the other hand, are either plus-end directed (most members of the kinesin superfamily) or minus-ended directed (dynein family and C-terminal kinesins). In recent years, a large number of ki- nesins and kinesin-likeproteins(KLPs) have been isolatedfrom vertebrates(Brady, 1985; Vale et al., 1985; Moore and Endow, 1996; Goldstein and Philp, 1999), invertebrates (Saxton et al., 1991), and fungi (McCaffry and Vale, 1989). The common fea- ture of all kinesins and KLPs is a conserved motor domain (,350 aa in length) with ATP- and MT-binding sites. Although most KLPs have their motor domains at the N terminus, some have their motor domains either at the C terminus or in the mid- dle of the protein. Beyond the motor domain, different KLPs show little or no sequence similarity (Bloom and Endow, 1994). Although much is known about kinesins and KLPs in ani- mals and yeast, little is known about these proteins in plants (Asada and Collings, 1997). The first indication of the presence of microtubule-dependent motors in plants came from the find- ing that phragmoplastmicrotubulesin tobacco BY-2 cells could translocate with respect to each other only in the presence of ATP or GTP (Asada et al., 1991). Subsequently, using a mon- Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado.