In Vitro Study of the Direct Effect of Microtubule Acetylation and Detyrosination on Kinesin Motility

Abstract

Kinesin-1 is a processive, plus end-directed motor that is a major component of microtubule-based intracellular transport. It has been previously shown through in vivo studies that kinesin-1 preferentially translocates along certain subsets of microtubules, which are marked with specific posttranslational modifications (PTMs). We hypothesize that PTMs of tubulin directly influence the interaction of kinesin-1 with microtubules. In our previous work, we concluded that kinesin-1 could not identify the enzymatic addition or removal of acetylation to K40 of α-tubulin since this modification did not directly affect its in vitro motility properties. In vivo, PTMs are rarely found in isolation of one another. In fact, there is an extensive overlap between acetylated and detyrosinated microtubules in vivo. Therefore we set out to directly test the influence of detyrosination on kinesin-1 motility. To obtain populations of tyrosinated and detyrosinated tubulin, we purified 99.5% tyrosinated tubulin from HeLa cells which was then used to generate detyrosinated microtubules by in vitro treatment with carboxypeptidase A. In order to examine the effect of microtubule detyrosination on kinesin -based transport, we characterized the single molecule motility properties of fluorescently labeled kinesin-1 on tyrosinated and detyrosinated microtubules using Total Internal Reflection Fluorescence (TIRF) microscopy. We observed that kinesin-1 shows enhanced binding to detyrosinated microtubules resulting in a marked difference in motility for the modified microtubules when compared with tyrosinated microtubules. Our results suggest that the exposure of a negatively charged glutamate residue upon detyrosination increases the electrostatic binding between the kinesin motor and the microtubule. We conclude that the kinesin-1 shows enhanced binding to detyrosinated microtubules and this may be an important molecular event underpinning the preferential transport by kinesin-1 observed in vivo.