Purification and Analysis of Authentic CLIP-170 and Recombinant Fragments*
- Jochen ScheeltOaFNbFNc,
- Philippe PierretOaFNcFNd,
- Janet E. RickardtOaFNcFNe,
- Georgios S. DiamantopoulostOa,
- Caterina ValettitOaFNf,
- F. Gisou van der GoottOg,
- Markus HänertOh,
- Ueli AebitOh and
- Thomas E. KreistOa,i
- From the tOaDepartment of Cell Biology, Sciences III, andtOgDepartment of Biochemistry, Sciences II, University of Geneva, CH-1211 Geneva 4 and tOhBiozentrum, M. E. Müller Institute for Structural Biology, University of Basel, CH-4056 Basel, Switzerland
Abstract
We have purified authentic CLIP-170 (cytoplasmic linker protein of170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is α-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced ∼37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubulesin vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.
Footnotes
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↵* This work was supported by the Swiss National Science Foundation (to T. E. K., J. E. R., F. G. v. d. G., and U. A.), the Canton de Genève (to T. E. K.), the M. E. Müller Foundation of Switzerland (to U. A), and the Canton Basel-Stadt (to M. H.).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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↵FNb Present address: Artemis Pharmaceuticals GmbH, Tübingen, Germany.
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↵FNc These authors contributed equally to this work.
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↵FNd Present address: Dept. of Cell Biology, Yale University School of Medicine, New Haven, CT.
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↵FNe Present address and to whom correspondence should be addressed: Dept. of Mental Health, University of Aberdeen Medical School, Foresterhill, Aberdeen, AB25 2ZD, Scotland. Tel.: 0044 1224 273101; Fax: 0044 1224 849191; E-mail: j.rickard@abdn.ac.uk.
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↵FNf Present address: Dept. of Experimental Medicine, University of Genova, Genova, Italy.
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↵i Deceased.
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↵2 G. S. Diamantopoulos, unpublished observations.
- Abbreviations:
- MBP
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microtubule-binding protein
- CLIP
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cytoplasmic linker protein
- mAb
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monoclonal antibody
- MAP
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microtubule-associated protein
- PAGE
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polyacrylamide gel electrophoresis
- PCR
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polymerase chain reaction
- PIPES
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1,4-piperazinediethanesulfonic acid
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- Received March 30, 1999.
- Revision received June 14, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
