Probing the kinesin-microtubule interaction.
Kinesin is a mechanoenzyme that couples adenosine triphosphate hydrolysis to the generation of force and movement along microtubules. To gain insight into the interactions of kinesin and microtubules, cross-linking, mapping, and proteolysis experiments were executed. The motor domain of kinesin was consistently cross-linked to both alpha- and beta-tubulin subunits. Initial mapping of the cross-linked kinesin suggested that amino acids within the N- and C-terminal cyanogen bromide fragments of the motor domain formed cross-links to both alpha- and beta-tubulin subunits. Mapping of the cross-linked tubulin suggested that cross-linking to kinesin motors occurred within the negatively charged, C-terminal cyanogen bromide fragments of alpha- and beta-tubulin subunits. Treatment of microtubules with subtilisin, a protease that cleaves C-terminal fragments from alpha- and beta-tubulin, reduced their ability to be cross-linked to kinesin motors supporting the idea that C-terminal sequences of alpha- and beta-tubulin may interact with kinesin motors. Finally, of three synthetic peptides, a peptide consisting of the last 12 C-terminal amino acids of beta-tubulin competitively interfered with the microtubule-stimulated adenosine triphosphatase activity of the kinesin motor, further suggesting that C-terminal sequences of beta-tubulin may be involved in kinesin binding.
Tucker
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Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0683, USA.
Goldstein
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LS
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018604:Epitope Mapping; D006863:Hydrogen-Ion Concentration; D016547:Kinesins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D013381:Subtilisins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D003432:Cross-Linking Reagents; D014404:Tubulin; D013381:Subtilisins; D016547:Kinesins