NCD activation of tubulin polymerization.
Tubulin dimer (tT) was purified from turkey erythrocytes. The motor domain of Drosophila non-claret disjunctional protein, NCD(335-700), was expressed in E. coli and purified. At 37 degrees C in the presence of GTP, the rate of polymerization of tT to microtubule (tMt) is accelerated over threefold by the presence of NCD(335-700). At 10 degrees C, the rate of tT polymerization is increased from zero, within experimental error, in the absence of NCD(335-700) to rates near those observed at 37 degrees C when NCD(335-700) is present. The NCD(335-700) concentration dependence of the rate indicated the reactive species was NCD(335-700)(n).tT, with n approximately 2. At 10 degrees C in the absence of GTP, polymerization does not occur, but tT activates NCD(335-700) MgATPase activity 10-fold. For the same conditions, using mians-NCD(335-700), which is modified with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid, the apparent K(D) for binding to tT is 2.3 x 10(-5) M in the presence of MgADP. Replacing ADP with AMPPNP or ATP has a negligible effect on K(D). Mians-NCD(335-700) binding to tMt is 10-fold stronger than to tT. The above data indicate NCD(335-700) binds at a functional site on tT. The stoichiometry is consistent with the formation of NCD(335-700)(2).tT which in vitro accelerates self-assembly initiation and/or polymerization by binding a second tT in a position favorable for tubulin-tubulin interaction. The data suggest that in vivo functional NCD binding to microtubule involves one motor domain binding to alpha- and beta-subunits at the interface of two different tubulin dimers in a protofilament.
Highsmith
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Department of Biochemistry, University of the Pacific, San Francisco, CA 94115, USA. shighsmith@sf.uop.edu
Thoene
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Sablin
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Polosukhina
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D000818:Animals; D001711:Biotransformation; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D004330:Drosophila; D029721:Drosophila Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014422:Turkeys
D029721:Drosophila Proteins; D014404:Tubulin; C066696:ncd protein, Drosophila; D006160:Guanosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D016547:Kinesins