ncd and kinesin motor domains interact with both alpha- and beta-tubulin.
Motor domains of the Drosophila minus-end-directed microtubule (MT) motor protein ncd, were found to saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to ncd, mixtures of ncd motor domain and MTs were treated with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide) (EDC). EDC treatment generated covalently cross-linked products of ncd and alpha-tubulin and of ncd and beta-tubulin, indicating that the ncd motor domain interacts with both alpha- and beta-tubulin. When the Drosophila kinesin motor domain protein was substituted for the ncd motor domain, cross-linked products of kinesin and alpha-tubulin and of kinesin and beta-tubulin were produced. EDC treatment of mixtures of ncd motor domain and unassembled tubulin dimers or of kinesin motor domain and unassembled tubulin dimers produced the same motor-tubulin products generated in the presence of MTs. These results indicate that kinesin family motors of opposite polarity interact with both tubulin monomers and support a model in which some portion of each protein's motor domain overlaps adjacent alpha- and beta-tubulin subunits.
Walker
R A
RA
Department of Biology, Virginia Polytechnic Institute, Blacksburg 24061-0406, USA.
D000818:Animals; D015153:Blotting, Western; D003432:Cross-Linking Reagents; D004330:Drosophila; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D016547:Kinesins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011994:Recombinant Proteins; D014404:Tubulin; C066696:ncd protein, Drosophila; D016547:Kinesins; D005022:Ethyldimethylaminopropyl Carbodiimide