Marya P K, Syed Z, Fraylich P E, Eagles P A
Department of Molecular Biology and Biophysics, Randall Institute, King's College, University of London, UK.
J Cell Sci. 1994 Jan;107 ( Pt 1):339-44. doi: 10.1242/jcs.107.1.339.
We have used a fluorescent derivative of kinesin, AF-kinesin (kinesin conjugated with 5-(iodoacetamido)fluorescein), to investigate the binding site of kinesin on microtubules and to compare this site with that to which tau binds. Microtubules saturated with tau will bind AF-kinesin in the presence of the ATP analogue, 5'-[beta,gamma-imino]triphosphate (AdoPP[NH]P). This shows that there are distinct binding sites for the two proteins. Further evidence comes from digestion studies where taxol-stabilised microtubules were treated with subtilisin, resulting in the cleavage of C-terminal residues from both the alpha- and beta-tubulin subunits. These treated microtubules can no longer bind tau, but are able to bind AF-kinesin in the presence of AdoPP[NH]P. Finally, AF-kinesin will support the gliding of subtilisin-digested microtubules in the presence of ATP at rates comparable to those obtained with non-digested microtubules. These results show directly that the binding site for kinesin is outside the C-terminal region of tubulin that is removed by subtilisin and is distinct from the binding site of tau.
我们使用了驱动蛋白的荧光衍生物AF-驱动蛋白（与5-（碘乙酰胺基）荧光素偶联的驱动蛋白），来研究驱动蛋白在微管上的结合位点，并将该位点与tau蛋白的结合位点进行比较。在ATP类似物5'-[β，γ-亚氨基]三磷酸（AdoPP[NH]P）存在的情况下，被tau蛋白饱和的微管会结合AF-驱动蛋白。这表明这两种蛋白质存在不同的结合位点。进一步的证据来自消化研究，用枯草杆菌蛋白酶处理紫杉醇稳定的微管，导致α-和β-微管蛋白亚基的C末端残基被切割。这些处理过的微管不再能结合tau蛋白，但在AdoPP[NH]P存在的情况下能够结合AF-驱动蛋白。最后，在ATP存在的情况下，AF-驱动蛋白将支持枯草杆菌蛋白酶消化的微管以与未消化微管相当的速率滑动。这些结果直接表明，驱动蛋白的结合位点在被枯草杆菌蛋白酶去除的微管蛋白C末端区域之外，且与tau蛋白的结合位点不同。
