Kinesin and tau bind to distinct sites on microtubules.

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.