Axoneme β-Tubulin Sequence Determines Attachment of Outer Dynein Arms

SummaryAxonemes of motile eukaryotic cilia and flagella have a conserved structure of nine doublet microtubules surrounding a central pair of microtubules. Outer and inner dynein arms on the doublets mediate axoneme motility [1]. Outer dynein arms (ODAs) attach to the doublets at specific interfaces 2, 3, 4 and 5. However, the molecular contacts of ODA-associated proteins with tubulins of the doublet microtubules are not known. We report here that attachment of ODAs requires glycine 56 in the β-tubulin internal variable region (IVR). We show that in Drosophila spermatogenesis, a single amino acid change at this position results in sperm axonemes markedly deficient in ODAs. Moreover, we found that axonemal β-tubulins throughout the phylogeny have invariant glycine 56 and a strongly conserved IVR, whereas nonaxonemal β-tubulins vary widely in IVR sequences. Our data reveal a deeply conserved physical requirement for assembly of the macromolecular architecture of the motile axoneme. Amino acid 56 projects into the microtubule lumen [6]. Imaging studies of axonemes indicate that several proteins may interact with the doublet-microtubule lumen 3, 4, 7 and 8. This region of β-tubulin may determine the conformation necessary for correct attachment of ODAs, or there may be sequence-specific interaction between β-tubulin and a protein involved in ODA attachment or stabilization.