Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic hela cells.
We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at approximately 30 microm/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at approximately 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of approximately 5 microm/min, and microtubules glide for an average of 3 microm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.
DeLuca
J G
JG
Department of Molecular, Cellular, and Developmental Biology and the Materials Research Laboratory, University of California, Santa Barbara, California 93106, USA.
Newton
C N
CN
Himes
R H
RH
Jordan
M A
MA
Wilson
L
L
D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000302:isolation & purification; D008870:Microtubules / Q000737:chemistry; D008938:Mitosis; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002868:Chromosomal Proteins, Non-Histone; C486566:KIFC1 protein, human; C068688:centromere protein E; D016547:Kinesins