Diffusion and directed movement: in vitro motile properties of fission yeast kinesin-14 Pkl1.
Fission yeast Pkl1 is a kinesin-14A family member that is known to be localized at the cellular spindle and is capable of hydrolyzing ATP. However, its motility has not been detected. Here, we show that Pkl1 is a slow, minus end-directed microtubule motor with a maximum velocity of 33+/-9 nm/s. The Km,MT value of steady-state ATPase activity of Pkl1 was as low as 6.4+/-1.1 nM, which is 20-30 times smaller than that of kinesin-1 and another kinesin-14A family member, Ncd, indicating a high affinity of Pkl1 for microtubules. However, the duty ratio of 0.05 indicates that Pkl1 spends only a small fraction of the ATPase cycle strongly associated with a microtubule. By using total internal reflection fluorescence microscopy, we demonstrated that single molecules of Pkl1 were not highly processive but only exhibited biased one-dimensional diffusion along microtubules, whereas several molecules of Pkl1, probably fewer than 10 molecules, cooperatively moved along microtubules and substantially reduced the diffusive component in the movement. Our results suggest that Pkl1 molecules work in groups to move and generate forces in a cooperative manner for their mitotic functions.
Furuta
Ken'ya
K
Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
Edamatsu
Masaki
M
Maeda
Yurina
Y
Toyoshima
Yoko Y
YY
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D015966:Gene Expression Regulation, Fungal; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D015513:Oncogene Proteins / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000378:metabolism*
D015513:Oncogene Proteins; D000251:Adenosine Triphosphatases; C502598:KIF14 protein, human; D016547:Kinesins