Biochemical Properties And Tissue Distribution Of Two Kinesin-like Proteins (KatB/KatC) In Arabidopsis

Kinesins are microtubule-based motor proteins that can move along microtubule (MT) using the energy from ATP hydrolysis. The proteins from animal cells have been biochemically characterized, universally showing an ATP-dependent MT-binding ability and a MT-stimulated ATPase activity that depend on the head motor domains. Kinesins are widely present in many eukaryotic cells and participate in various types of intracellular procession, such as cell morphogenesis, organelle transport, cell division, nuclear migration, mRNA localization, and signal transduction. There are sixty-one kinesin isoforms in Arabidopsis, KatB and KatC are two C-teminal motor proteins and they share 84% amino acid identity. KatB and KatC were cloned early in 1993, but little is known about their biochemical characteristics and functions. Revealing the biochemical properties of KatB and KatC in vitro and their tissue distributions in vivo is significant for understanding the roles of plant kinesin isoforms.The N-terminal tail of KatC heavy chain (1-128 aa, KatC128) was expressed in E.cloi and purified by Ni affinity chromatography. The cosedimentation assay shows that the N-terminal tail of KatC can bind on MTs at the ATP-indepenent manner. CCD microscopy observation indicates that this region is not able to induce MT into bundles. To identify the region responsible for ATP-independent MT interaction of KatC 128, 6 cDNAs coding various regions of KatC tail regions fused with His-tag were expressed in E.cloi and affinity-purified. Purified proteins were used in MT cosedimentation assay, showing that the polypeptide of 73-86aa is responsible for MT binding, and different deletion polypeptides have different MT binding ration that may result from the conformation-dependent. Arg77 and Arg79 were substituted to Val respecitively for making site-directed mutagenesis. The results show that Arg77 and Arg79 are essential for KatC 128 binding to microtubules. In addition, we expressed and purified the region of KatB lacking the motor domain. Cosedimendation assay indicates that KatB tail region also can bind onto MTs. KatB (l-383aa) and KatC (l-394aa) with stalk region were fused to GST and His tag respectively, pull down results show that KatB and KatC not only can form homodimer byself but also form heterodimer between them.Using gus as report gene, the specific expression patterns of the two promoters of KatB and KatC were analyzed. Gus gene was expressed in root meristematic zone, stele and lateral root under the control of katC's promoter, by contrast, gus activity driving by KatC promoter is observed in root meristematic zone and young hair. In addention, KatC was expressed in silique, while KatB was expressed widely in leaf primordium, leaf vein, trichome and anther, besides silique.In order to understand the expression of KatB and KatC in Arabidopsis, antiserums against the specific polypetides of KatB and KatC were prepared, purified and used to analyze the expression levels of KatB and KatC in stem, root, leaf, flower, and slique based on western blot. KatB signal was detected in all the tissues except slique, while a weak KatC signal in leaf and flower and a clear sigal in silique.To reveal the functions of katB and katC genes in Arabidopsis, single T-DNA insertion mutants and double mutants of katB and katC were obtained by PCR and RT-PCR, and no obvious abnormal phenotypes were observed during the growth and development of the mutant plants.