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Reversal in the direction of movement of a molecular motor

Nature volume 389, pages 93–96 (1997)Cite this article

Kinesin and non-claret disjunctional (ncd) are molecular motors of the kinesin superfamily that move in opposite directions along microtubules. The molecular basis underlying the direction of movement is unclear, although it is thought to be an intrinsic property of the motor domain, a conserved region about 330 amino acids in length1,2,3. The motor domain is found at the amino terminus in conventional kinesins, but at the carboxy terminus in ncd4,5. Here we report on a chimaera composed of the motor domain of the minus-end-directed kinesin of Neurospora crassa6. The bacterially expressed fusion protein was tested in motility assays using polarity-marked microtubules7. Surprisingly, the chimaera moved towards the plus end, demonstrating that the polarity of force generation of the ncd motor domain has been reversed. This finding indicates that the domain organization, particularly the position of the motor domain, is of fundamental importance for the polarity of force production. It also demonstrates that the direction of microtubule movement is not controlled solely by the motor domain8.

The identity of this polypeptide was investigated in immunoblots using a polyclonal antibody against the Nkin stalk/tail domain6 and a peptide antibody (MMR48) against a consensus sequence 24 amino acids in length in the HIPYR region of animal kinesins13. The Nkin antibody recognized Nkin as well as the chimaera, but not GST–MC5 (ncd). Antibody MMR48 reacted strongly with Nkin, which was to be expected given the high degree of sequence conservation of the HIPYR peptide between animal kinesins and Nkin. Its reaction with GST–MC5, however, was much weaker, because of considerable sequence divergence in the HIPYR peptide between ncd and animal kinesins. The chimaera showed the same weak reaction with MMR48 as GST–MC5. Thus the chimaera combines the immunological characteristics of the Nkin stalk/tail domain and the ncd motor domain.

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Figure 1: Overview of the domain organization of the non-claret disjunctional motor (ncd), Neurospora kinesin (Nkin), and the chimaera (ncd.
Figure 2: a, Isolation of the ncd–Nkin chimaera expressed in bacteria.
Figure 3: Directionality of ncd–Nkin motor activity using polarity-marked microtubules.

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Acknowledgements

We thank S. Endow for the GST–MC5 clone; M. McNiven for the MMR48 antibody; M. Fritz for the rhodamine-labelled tubulin; U. Euteneuer, H. Felgner, R. Gräf and S. Linder for advice; and S. Fuchs for technical assistance. This work was supported by a Graduiertenkolleg fellowship to U.H. and a grant from the DFG to M.S.

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  1. Adolf-Butenandt-Institut, Zellbiologie, University of Munich, Schillerstrasse 42, Munich, 80336, Germany

    Ulrike Henningsen & Manfred Schliwa

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Correspondence to Manfred Schliwa.

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Henningsen, U., Schliwa, M. Reversal in the direction of movement of a molecular motor. Nature 389, 93–96 (1997). https://doi.org/10.1038/38022

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