Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor

Abstract

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 μm/s at 37°C and 0.24 μm/s at 25°C. The motor activity copurified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874 000 Da and estimated dimensions of 2 mm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.