PROBING THE KINESIN-MICROTUBULE INTERACTION

Kinesin is a mechanoenzyme that couples adenosine triphosphate hydroly sis to the generation of force and movement along microtubules. To gai n insight into the interactions of kinesin and microtubules, cross-lin king, mapping, and proteolysis experiments were executed. The motor do main of kinesin was consistently crosslinked to both alpha- and beta-t ubulin subunits. Initial mapping of the cross-linked kinesin suggested that amino acids within the N- and C-terminal cyanogen bromide fragme nts of the motor domain formed cross-links to both alpha- and beta-tub ulin subunits, Mapping of the cross-linked tubulin suggested that cros s-linking to kinesin motors occurred within the negatively charged, C- terminal cyanogen bromide fragments of alpha- and beta-tubulin subunit s. Treatment of microtubules with subtilisin, a protease that cleaves C-terminal fragments from alpha- and beta-tubulin, reduced their abili ty to be cross-linked to kinesin motors supporting the idea that C-ter minal sequences of alpha- and beta-tubulin may interact with kinesin m otors. Finally, of three synthetic peptides, a peptide consisting of t he last 12 C-terminal amino acids of beta-tubulin competitively interf ered 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.