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Cotton GhKCH2, a Plant-specific Kinesin, is Low-affinitive and Nucleotide-independent as Binding to Microtubule

  • Xu, Tao (State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University) ;
  • Sun, Xuewei (State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University) ;
  • Jiang, Shiling (State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University) ;
  • Ren, Dongtao (State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University) ;
  • Liu, Guoqin (State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University)
  • Published : 2007.09.30
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Abstract

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

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References

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