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

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 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co‐purified 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 nm 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.