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Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin - PubMed

  • ️Sun Jan 01 2006

Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin

Seiichi Uchimura et al. EMBO J. 2006.

Abstract

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.

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Figures

Figure 1
Figure 1

Design of the H12 mutants of β-tubulin in Saccharomyces cerevisiae. (A) Sequences of the H12 region are shown with negatively charged residues indicated by blue and the residues substituted by alanines are indicated by red. (B) A ribbon diagram of the tubulin dimer viewed from the side of the microtubule with its minus end to the left (Nogales et al, 1998). Image analysis of the kinesin–microtubule complex revealed that in both nucleotide free and AMP-PNP state, kinesin motor domain is associated in close proximity to H11 (orange), H12 (cyan), and the COOH terminus (undefined in crystal structure) of β-tubulin (Kikkawa et al, 2000; Hoenger et al, 2000). The acidic residues in H12 mutagenized to alanine are indicated in blue.

Figure 2
Figure 2

Purity of yeast tubulin and images of polymerized microtubules. (A) SDS–PAGE analysis of purified tubulin. Lane 1, yeast wild-type tubulin; lane 2, E410A; lane 3, E412A; lane 4, D417A; lane 5, E421A; and lane 6, porcine brain tubulin. In each lane, 1 μg of sample was loaded and stained by Coomassie blue. In SDS gel containing Sigma SDS (L-5750), α- and β-polypeptide of porcine brain tubulin were separated more as compared to these peptides purified from yeast cells (Best et al, 1981; Bode et al, 2003). (B) Dark-field images of the microtubules polymerized from yeast wild-type and mutated tubulins in the presence of 1 μM Taxol. Bar=5 μm.

Figure 3
Figure 3

Motility assays using mutated microtubules. (A) In microtubule gliding assay, the fraction of microtubules that moved a distance greater than their own length (∼3 μm), f, was measured, and (B) plotted as a function of kinesin density, ρ. Total number of microtubules counted for wild-type (black), E410A (blue), E412A (orange), D417A (red), and E421A (green) were 495, 328, 657, 496, and 388, respectively. Continuous curves are the plot of equation (1). Error bars represent the statistical counting errors, calculated according to Materials and methods. (C) In single molecule motility assay using TIRFM, the motility of HK560-Cy3 was examined, and (D) the distribution of the kinesin run length and the velocity was analyzed for both the wild-type and the mutated microtubules. HK560-Cy3 did not interact with the E410A and D417A microtubules. Total number of the events counted for wild-type, E412A, and E421A were 374, 329, and 333, respectively.The mean run length and the mean velocity, calculated according to Supplementary Methods, are shown in each panel with the errors of the curve fits.

Figure 4
Figure 4

Unbinding force distribution of one-headed kinesin in (A) ADP and (B) AMP-PNP state. An external load was applied towards either the plus end (orange for (A) and red for (B)) or the minus end of the microtubule (light and dark green for (A) and (B), respectively). The stiffness of the trap was 0.038 pN/nm (ADP) and 0.076 pN/nm (AMP-PNP). The average unbinding force (pN) with s.e.m. is shown in each panel.

Figure 5
Figure 5

Stall force measurement. (A) Representative tracing of a trapped bead powered by a conventional two-headed kinesin along the wild-type, E410A, D417A, and E421A microtubules, measured at the trap stiffness of 0.076 pN/nm. Light shaded, unfiltered; solid, filtered at 100 Hz. (B) Distribution of the stall force for wild-type and mutated microtubules. The average stall force with s.e.m. is shown in each panel. Total number of events counted are (from top to bottom) 61, 104, 102, and 69, respectively. (C) The stall force plotted against the unbinding force for minus- (left) and plus-end loading (right). The stall force was linearly related to the unbinding force for minus-end loading (linear coefficient; 0.73).

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