Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and har-bor various post-translational modifications. However, how tubulin isotypes determine the activities of reg-ulators for microtubule stability and modifications remains unknown. Here, we show that human a4A-tubulin, a conserved genetically detyrosinated a-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based in vitro assays. The incorporation of a4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of a-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of a-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.