Tubulin assembly probed with antibodies to synthetic peptides.
Antibodies to synthetic peptides from the alpha and beta-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 alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(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 alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(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 alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.
Centro de Investigaciones Biológicas, C.S.I.C., Madrid, Spain.
M A
MA
Arévalo
J M
JM
Nieto
D
D
Andreu
J M
JM
Andreu
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D008274:Magnesium; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D017239:Paclitaxel; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D010447:Peptide Hydrolases; D014404:Tubulin / Q000378:metabolism*; D015032:Zinc
D000470:Alkaloids; D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D014404:Tubulin; D010447:Peptide Hydrolases; D008274:Magnesium; D015032:Zinc; D017239:Paclitaxel