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© The Rockefeller University Press, 0021-9525/1998//1407 $5.00
The Journal of Cell Biology, Volume 140, Number 6, , 1998 1407-1416

Article

Role of the Kinesin Neck Region in Processive Microtubule-based Motility



Laura Romberg*, Daniel W. Pierce*,{ddagger}, and Ronald D. Vale{ddagger},*

* Departments of Cellular and Molecular Pharmacology, and Departments of Biochemistry and Biophysics, University of California, San Francisco, California 94143; and {ddagger} Howard Hughes Medical Institute, San Francisco, CA 94143

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin–GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.

Abbreviations used in this paper: aa, amino acid; GFP, green fluorescent protein.

Address all correspondence to Ron Vale, Department of Cellular and Molecular Pharmacology, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143. Tel.: (415) 476-6380. Fax: (415) 476-5233. E-mail: vale{at}phy.ucsf.edu


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