INTERACTION OF MICROTUBULE-ASSOCIATED PROTEINS WITH MICROTUBULES - YEAST LYSYL-TRANSFER RNA AND VALYL-TRANSFER RNA-SYNTHETASES AND TAU-218-235 SYNTHETIC PEPTIDE AS MODEL SYSTEMS

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS; KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau-protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a K(D) in the 10(-6) M range, and tau 218-235 binds with a K(D) in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. Tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha-beta(S) tubulin and 1/alpha(S)beta(S) tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.