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

Seiichi Uchimura, Yusuke Oguchi, Miho Katsuki, Takeo Usui, Hiroyuki Osada, Jun Ichi Nikawa, Shin'ichi Ishiwata, Etsuko Muto

    Research output: Contribution to journalArticle

    36 Citations (Scopus)

    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 α-helix 12 of β-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′-[β,γ-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.

    Original languageEnglish
    Pages (from-to)5932-5941
    Number of pages10
    JournalEMBO Journal
    Volume25
    Issue number24
    DOIs
    Publication statusPublished - 2006 Dec 13

    Fingerprint

    Kinesin
    Tubulin
    Microtubules
    Binding Sites
    Adenylyl Imidodiphosphate
    Saccharomycetales
    Force measurement
    Alanine
    Yeast
    Adenosine
    Assays

    Keywords

    • Kinesin
    • Microtubule
    • Mutant analysis
    • Stall force
    • Yeast

    ASJC Scopus subject areas

    • Genetics
    • Cell Biology

    Cite this

    Uchimura, S., Oguchi, Y., Katsuki, M., Usui, T., Osada, H., Nikawa, J. I., ... Muto, E. (2006). Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. EMBO Journal, 25(24), 5932-5941. https://doi.org/10.1038/sj.emboj.7601442

    Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. / Uchimura, Seiichi; Oguchi, Yusuke; Katsuki, Miho; Usui, Takeo; Osada, Hiroyuki; Nikawa, Jun Ichi; Ishiwata, Shin'ichi; Muto, Etsuko.

    In: EMBO Journal, Vol. 25, No. 24, 13.12.2006, p. 5932-5941.

    Research output: Contribution to journalArticle

    Uchimura, S, Oguchi, Y, Katsuki, M, Usui, T, Osada, H, Nikawa, JI, Ishiwata, S & Muto, E 2006, 'Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin', EMBO Journal, vol. 25, no. 24, pp. 5932-5941. https://doi.org/10.1038/sj.emboj.7601442
    Uchimura, Seiichi ; Oguchi, Yusuke ; Katsuki, Miho ; Usui, Takeo ; Osada, Hiroyuki ; Nikawa, Jun Ichi ; Ishiwata, Shin'ichi ; Muto, Etsuko. / Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. In: EMBO Journal. 2006 ; Vol. 25, No. 24. pp. 5932-5941.
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