TUBULIN ASSEMBLY PROBED WITH ANTIBODIES TO SYNTHETIC PEPTIDES

Antibodies to synthetic peptides from the α and β-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin αβ heterodimer. IgG antibodies to the α(430-443) and β(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2+-induced tubulin sheets, Mg2+-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions α(155-168) did not react with microtubules, while the equivalent zone β(153-165) was accessible. The α(214-226) and β(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2+-induced sheets and Mg2+-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions α(214-226) and β(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions α(241-256) and β(214-226) are suggested to be related to the α-β binding interface within the heterodimer. © 1990 Academic Press Limited.