Acetylation of Alpha Tubulin Lysine-40 Alone is not Sufficient for Changes in Kinesin-1 Motility

Abstract

Kinesin-1 is a processive, plus end-directed motor that is responsible for major 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 order to investigate the role of acetylation of α-tubulin at Lysine 40 (K40) in this context, we examined the binding affinity of kinesin in solution to acetylated and deacetylated microtubules in the presence of AMPPNP. We further characterized the single molecule motility properties of kinesin-1 on acetylated and deacetylated microtubules using Total Internal Reflection Fluorescence (TIRF) microscopy. To generate acetylated and deacetyalated microtubules, purified bovine tubulin was treated with the enzymes MEC-17 and SIRT2, respectively. Kinesin-1 motors were either expressed in COS cells or purified from bacterial cells. We found that kinesin-1 shows similar binding affinity, velocity and run length on acetylated and deacetylated microtubules as measured in these in vitro assays. Our results suggest that an alteration in the state of acetylation of K40 on α-tubulin in the microtubules does not result in changes in the catalytic cycle and strong or weak- binding states of the motor. We conclude that kinesin-1 cannot directly recognize the presence of an acetyl group on K40 of α-tubulin and hence this modification alone is not sufficient to explain the preferential motility of kinesin-1 observed in vivo. Rather, K40 acetylation appears to mark a subset of microtubules with other structural or biochemical alterations that are recognized as trafficking cues by kinesin-1.