The human chromokinesin Kid is a plus end-directed microtubule-based motor

Abstract

Kid is a kinesin-like DNA-binding protein known to be involved in chromosome movement during mitosis, although its actual motor function has not been demonstrated. Here, we describe the initial characterization of Kid as a microtubule-based motor using optical trapping microscopy. A bacterially expressed fusion protein consisting of a truncated Kid fragment (amino acids 1-388 or 1-439) is indeed an active microtubule motor with an average speed of approximately 160 nm/s, and the polarity of movement is plus end directed. We could not detect processive movement of either monomeric Kid or dimerizing chimeric Kid; however, low levels of processivity (a few steps) cannot be detected with our method. These results are consistent with Kid having a role in chromosome congression in vivo, where it would be responsible for the polar ejection forces acting on the chromosome arms.

Fig. 1. Kid constructs used in this study. (A) Schematic representation of the Kid and chimera constructs compared with full-length Kid protein. The numbers show amino acid positions. MOTOR, kinesin-like motor domain including ATP hydrolysis site and microtubule-binding sites; P, cdc2 phosphorylation consensus site; COIL, predicted coiled-coil domain; HhH1, predicted helix–hairpin–helix domain; kin-COIL, kinesin’s coiled-coil domain; BCCP, biotin carboxyl carrier protein. (B) The expressed, purified proteins. Left: Coomassie Blue staining. Molecular weight markers (×10³) are indicated on the left. Right: western blot probed with the anti-biotin antibody.

Fig. 2. Sedimentation profiles of motor proteins by sucrose density gradient centrifugation. Open squares, kin351–BCCP; filled squares, kin410–BCCP; filled circles, Kid388–BCCP; open circles, Kid439– BCCP; filled triangles, Kidsin–BCCP. The relative concentrations of motor proteins in each fraction were estimated from scans of Coomassie Blue-stained SDS–polyacrylamide gels. The peaks of Kid388–BCCP and Kid439–BCCP were in similar positions to the peak of kin351–BCCP used as a monomer control. Both sedimented at a lower mass position than the BSA standard peak at fraction number 8. The peak of Kidsin–BCCP was in the same position as kin410–BCCP, the dimer control, and at a higher mass position than that of BSA.

Fig. 3. Bead displacement driven by motor proteins within an optical trap. (A) Kid439–BCCP, (B) kin410–BCCP and (C) BCCP–MC1. The traces refer to displacements along the microtubule axis, and plus endward displacements are positive. The trap stiffness was 0.019 pN/nm.

Fig. 4. Displacement of the bead coated with two kinds of motor proteins. (A) Kin410–BCCP and BCCP-MC1 (1:10), (B) Kid439–BCCP and BCCP–MC1 (1:1), (C) Kid439–BCCP and kin410–BCCP (10:1). The molar ratios of the two proteins in solution before binding to the beads are in parentheses. The trap stiffness was 0.022 pN/nm.

Fig. 5. Probability of moving and binding to beads at various molar ratios of four kinds of motor proteins at mixing. Filled squares and open circles denote the fraction of beads binding in 1 mM AMPPNP and moving in 1 mM ATP, respectively. (A) Kid439–BCCP, (B) Kidsin–BCCP and (C) BCCP-MC1. The plots are fitted to 1 – exp(–x/b) for binding beads (broken line) and 1 – (–x/m)exp(–x/m) for moving beads (dotted line), where x is the molar ratio of motor protein to beads following mixing, and b and m are the respective fitting parameters; b and m for Kid439–BCCP are 1849 and 3054, for Kidsin–BCCP are 2302 and 3622, and for BCCP–MC1 are 1643 and 3280. (D) Kin410–BCCP; the plots are fitted to 1 – exp(–x/2039) for binding beads (broken line) and 1 – exp(–x/3250) for moving beads (solid line).