Subcellular Localization Of A Kinesin-Like Protein (AtKP1) In Arabidopsis

The microtubule motor protein kinesin was identified by Vale in 1985 as the motile force underlying movement of particles along the microtubules of the giant axon of the squid. Kinesin was shown to be capable of binding to microtubules and, in the presence of ATP, of moving along microtubules, which enable the motors to perform essential roles in vesicle and organelle transport, spindle function and chromosome motility, and regulation of microtubule dynamics.Recently, a new kinesin gene AtKP1 (GenBank accession no. AF398149) , was cloned from Arabidopsis thaliana by Haiqing Wang in our lab. Sequence analysis of AtKP1 cDNA revealed an open reading frame of 3264bp, encoding a protein of 1087 amino acids. The basic biochemical characteristics of AtKP1 motor domain (300-802aa) were analyzed, and its anti-serum against AtKP1 specific polypetides (886-1087aa) was prepared.In this paper, we purified the AtKP1 antibody using ionexchage chromatograph column (DEAE-Cellulase52) and affinity purification slurry (Ni-NTA), and analyzed its specificity. The protein extract of whole Arabidopsis shoots was prepared, seperated by SDS-PAGE and immunoblotted with preserum, anti-serum and affinity purified anti-AtKP1 antibody, respectively. Only one immunoreactive signal with an M, of 125KD was obtained with both anti-serum and affinity-purified anti-AtKP1, which was not recognized by preserum. To verify that the polypeptide recognized by the antibody was AtKP1, we prepared the E.coli lyates containing the histidine-tagged AtKP1 specific polypeptide (886-1087aa) or entire AtKP1. When subjected to affinity purified antibody, monoclonal anti-Histidine tag antibody and monoclonal anti-animal-kinesin antibody, respectively, both lyates produced a single immunoreactive band with an M, of 28KD or 125KD, indicating that the 125 KD polypeptide recognized by affinity-purified antibody was our target protein, AtKP1.The expression pattern of AtKP1 protein in organs of Arabidopsis was examined by both immunoblot and Immunofluorescence analysis with affinity-purified anti-AtKP1 antibody. For Western blot, the relative amount of total protein from each organ was determined using the relative amount of the β-actin as a reference. The results showed that AtKP1 was the highest enriched in flower, and then in stems, leaves and roots in turn. Immunofluorescence signals could be obtained in root meristematic cones, lateral roots and leaves.Using the affinity-purified anti-AtKP1 antibody and anti-tublin antibody, we co-localized AtKP1 and microtubules (MTs) in intact Arabidopsis root tip cells at different stages of cell division. In interphase cells, AtKP1 was present throughout the cytoplasm. In preprophase cells, AtKP1 still localized to particle-like structures. In metaphase cells, no AtKP1 was detected on spindle microtubules. When microtubules concentrated to form phragnoplast, AtKP1 still keptstaying throughout the cytoplasm. The results suggested that AtKP1 associated with organelles and/or vesicles in Arabidopsis cells.In order to acquire the detail localization of AtKP1, we seperated subcellular fractions by sucrose density gradient ultracentrifugation. We obtained the mitochondria, cytosol, microsome and chloroplast fractions from arabidopsis leaves and probed them in immunoblotting with anti-AtKP1 antibody. The results indicated that only the mitochondria fractions contained AtKP1 which could not be released by exposure to high-salt solution.As a conclusion, this study demonstrated that the kinesin-like protein, AtKP1, differently from most of other higher plant kinesins and kinesin-like proteins, was not involved in cell division, but tightly bound to mitochondria in arabidopsis cells, and might be involved in the acitivity of mitochondria.