The Kinesin-related Protein Kip1p of Saccharomyces cerevisiae Is Bipolar*
- From the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085
Abstract
Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM50884 (to D. M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. Tel.: 215-573-3636; Fax: 215-573-5851; E-mail: roof@mail.med.upenn.edu.
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↵2 D. M. Gordon and D. M. Roof, manuscript in preparation.
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↵3 D. M. Roof, unpublished data.
- Abbreviations:
- HA
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hemagglutinin
- His6
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a tag of six consecutive histidine residues
- Ni-NTA agarose
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Ni2+-nitriloacetic acid agarose
- GST
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glutathioneS-transferase
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- Received June 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
