Microtubule assembly protects the region 28-38 of the beta-tubulin subunit.

Abstract

Polyclonal antibodies have been raised against the peptide 28-38 of the beta-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all beta-tubulin subunits, except for beta'-tubulin isotypes, and did not recognize the alpha-tubulin subunit. The 28-38 region does not play a role in the interaction between the alpha- and beta-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubular associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of beta-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the beta-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly.