Two kinesins from Arabidopsis, KatB and KatC, have a second microtubule-binding site in the tail domain.
Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubulebinding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.
Jiang
Shiling
S
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Li
Ming
M
Xu
Tao
T
Ren
Dongtao
D
Liu
Guoqin
G
D000255:Adenosine Triphosphate / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; C089152:KatB protein, Arabidopsis; C089153:KatC protein, Arabidopsis; D016547:Kinesins