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.
Thrower
D A
DA
Department of Biological Sciences, University of California, Santa Barbara 93106.
Jordan
M A
MA
Schaar
B T
BT
Yen
T J
TJ
Wilson
L
L
D002465:Cell Movement / Q000187:drug effects; D002848:Chromatography, DEAE-Cellulose; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D008677:Metaphase; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology; D008938:Mitosis; D008970:Molecular Weight; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology
D002868:Chromosomal Proteins, Non-Histone; D003854:Deoxyribonucleotides; C068688:centromere protein E; D014638:Vanadates; D004398:Dyneins; D016547:Kinesins