Differential modification of the C-terminal tails of different α-tubulins and their importance for microtubule function in vivo

Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and β-tubulin. However, very little is known about the specific modifications found on the different tubulin isotypes in vivo and the role of these PTMs in MT transport and other cellular processes in vivo. In this study, we found that in Drosophila ovaries, glutamylation of α-tubulin isotypes occurred clearly on the C-terminal ends of αTub84B and αTub84D (αTub84B/D). In contrast, the ovarian α-tubulin, αTub67C, is not glutamylated. The C-terminal ends of αTub84B/D are glutamylated at several glutamyl side chains in various combinations. Drosophila TTLL5 is required for the mono- and poly-glutamylation of ovarian αTub84B/D and with this for the proper localization of glutamylated microtubules. Similarly, proper kinesin distribution in the germline also depends on TTLL5 as well as the refining of Staufen localization and the normal fast ooplasmic streaming with its directional movement, two processes known to depend on kinesin activities. In the nervous system, the pausing of anterograde axonal transport of mitochondria is affected by an enzymatically dead mutant of TTLL5. Our results demonstrate in vivo roles of TTLL5 in differential glutamylation of α-tubulins and point to the in vivo importance of α-tubulin glutamylation for kinesin-dependent processes. Summary α-tubulin glutamylation was established in the C-terminal domain of Drosophila αTub84B and αTub84D (αTub84B/D). Multiple glutamyl residues were pinpointed in this domain. The female germline α-tubulin, αTub67C, is, however, not glutamylated. TTLL5 is required for mono- and poly-glutamylation of αTub84B/D. TTLL5 is required for the proper Kinesin heavy chain (Khc) distribution in the germline, and the kinesin-based refinement of Staufen localization and ooplasmic streaming during late oogenesis. TTLL5 is needed for the pausing of anterograde trafficking of mitochondria in axons.