Reconstitution of an active human CENP-E motor

Abstract

CENP-E is a large kinesin motor protein which plays pivotal roles in mitosis by facilitating chromosome capture and alignment, and promoting microtubule flux in the spindle. So far, it has not been possible to obtain active human CENP-E to study its molecular properties. Xenopus CENP-E motor has been characterized in vitro and is used as a model motor; however, its protein sequence differs significantly from human CENP-E. Here, we characterize human CENP-E motility in vitro. Full-length CENP-E exhibits an increase in run length and longer residency times on microtubules when compared to CENP-E motor truncations, indicating that the C-terminal microtubulebinding site enhances the processivity when the full-length motor is active. In contrast with constitutively active human CENP-E truncations, fulllength human CENP-E has a reduced microtubule landing rate in vitro, suggesting that the non-motor coiled-coil regions self-regulate motor activity. Together, we demonstrate that human CENP-E is a processive motor, providing a useful tool to study the mechanistic basis for how human CENP-E drives chromosome congression and spindle organization during human cell division.