AT LEAST ONE OF THE PROTOFILAMENTS IN FLAGELLAR MICROTUBULES IS NOT COMPOSED OF TUBULIN

Background: The core of the eukaryotic flagellum is the axoneme, a com plex motile organelle composed of similar to 200 different polypeptide s. The most prominent components of the axoneme are the central pair a nd nine outer doublet microtubules. Each doublet microtubule contains an A and a B tubule; these are composed, respectively, of 13 and 10-11 protofilaments, all of which are thought to be made of tubulin. The m echanisms that control the assembly of the doublet microtubules and es tablish the periodic spacings of associated proteins, such as dynein a rms and radial spokes, are unknown. Tektins, a set of microtubule-asso ciated proteins, are present in the axoneme as stable filaments that r emain after the extraction of doubler microtubules; they are localized near to where the B tubule attaches to the A tubule and near to the b inding sites for radial spokes, inner dynein arms and nexin links. Tek tin filaments may contribute in an interesting way to the structural p roperties of axonemes. Results: We have fractionated doublet microtubu les from sea urchin sperm flagella into ribbons of stable protofilamen ts, which can be shown to originate from the A tubule. Using cryo-elec tron microscopy, conventional electron microscopy, scanning transmissi on electron microscopy, three-dimensional reconstruction and kinesin d ecoration, we have found that one protofilament in the ribbon is not c omposed of tubulin. This protofilament is an integral protofilament of the A tubule wall, has less mass per unit length than tubulin and doe s not bind kinesin. Conclusion: Contrary to what is generally assumed, at least one protofilament in the wall of the A tubule is not compose d of tubulin. Our data suggest that this non-tubulin protofilament is primarily composed of tektins, proteins that show some structural simi larity to intermediate filament proteins. A 480 A axial periodicity wi thin these ribbons, revealed by scanning transmission electron microsc opy, can be related to the structure of tektin, and may determine the large-scale structure of the axoneme in terms of the binding of dynein , nexin and radial spokes to the doublet microtubule.