The carboxy-terminal sequence Asp427-Glu432 of beta-tubulin plays an important function in axonemal motility

Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression o f several isogenes and from the existence of various post-translational mod ifications. In order to characterize tubulin isoforms and tubulin domains t hat are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea-urchin sperm tails. The monoclona l antibodies obtained were screened for their potency to inhibit demembrana ted-reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specif ically with a subset of P-tubulin isoforms. Limited proteolysis, HPLC, pept ide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423-Glu 435 of the C-terminal domain of Lytechinus pictus beta 2-tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immun oblotting analysis further narrowed the amino acids important for antibody recognition to Asp427-Glu432. Because the primary effect of this antibody o n sperm motility is to decrease the flagellar beat frequency, we suggest th at this sequence is involved in the tubulin-dynein head interaction.