The Effects of Removal of C-termini of Tubulin for Mitotic Kinesin CENP-E Microtubule Interactions

Abstract

The carboxyl-terminus of both a- and b- tubulin consists of helices H11 and H12 and are located on the outer surface of the microtubule (MT). H12 extends into a disordered region that is characterized by a highly negatively charged motif of approximately 10-18 residues (CTT). These MT CTTs have been shown to affect the MT interactions with kinesin molecular motors for function. The research presented here tests the hypothesis that the MT CTTs modulate the behavior of the kinesin motor CENP-E with the MT and affect ATP-promoted motility. Subtilisin was used to remove the CTTs of a,b-tubulin, and the impact of the loss of the CTTs was evaluated by MT-CENP-E cosedimentation and CENP-E promoted MT gliding assays. The cosedimentation results show a dramatic decrease in MT affinity to subtilisin-MTs in the presence of 1 mM MgAMPPNP although 100% CENP-E binding is achieved. In contrast, the 1 mM MgADP results demonstrate an increase in CENP-E binding to subtilisin-MTs and an increase in MT affinity in comparison to native MTs. The in vitro motility assays reveal an increase in the rate of CENP-E promoted MT gliding on subtilisin MTs at 7.2 nm/s relative to 5.1 nm/s on native MTs. However, after 15 min, most subtilisin-MTs detached from the coverslip suggesting almost complete dissociation of the subtilisin-MT-CENP-E complex. In contrast, CENP-E promoted native MT gliding for >60 min. This study provides further evidence for the importance of the electrostatic interactions mediated by the MT CTTs for CENP-E mitotic function.