Tubulin engineering by semisynthesis reveals that polyglutamylation directs detyrosination

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here, we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails, which are site-specifically glutamylated, to recombinant human tubulin heterodimers applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/SVBP in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.

微管（Microtubules）是细胞骨架的关键组成成分，其携带的翻译后修饰（post-translational modifications, PTMs）对于调控关键细胞过程具有重要意义。尤其在神经元中，长寿命微管同时存在α-微管蛋白的去酪氨酸化与多谷氨酰化修饰。此类翻译后修饰的失调可引发发育缺陷与神经退行性疾病。由于缺乏研究这类修饰的调控与功能的工具，调控此类修饰模式的分子机制尚未得到充分阐释。本研究采用分选酶（sortase）与内含肽（intein）介导的串联转酰胺化策略，将经位点特异性谷氨酰化修饰的合成α-微管蛋白尾巴连接至重组人微管蛋白异二聚体，获得了在C端尾巴带有精准定义修饰的全功能微管蛋白。利用这些定制化微管蛋白重构的微管，我们发现α-微管蛋白的多谷氨酰化可通过增强微管酪氨酸羧肽酶vasohibin/SVBP的活性，促进其去酪氨酸化，且该调控过程依赖于多谷氨酰链的长度。此外，我们在细胞中调控多谷氨酰化水平时，观察到去酪氨酸化程度也发生相应变化，证实了去酪氨酸化循环与多谷氨酰化之间的关联。