Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules

Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FANS, expressed it in Escherichia coil cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FANS was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of beta-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing D-tuhulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 hinds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.