Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell-substrate adhesion were significantly compromised in cultured αTat1(-/-) fibroblasts. First, αTat1(-/-) fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1(-/-) cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1(-/-) cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.

Protein acetylation is an important posttranslational modification with the recent identification of new substrates and enzymes, new links to disease, and modulators of protein acetylation for therapy. α-Tubulin acetyltransferase (αTAT1) is the major α-tubulin lysine-40 (K40) acetyltransferase in mammals, nematodes, and protozoa, and its activity plays a conserved role in several microtubule-based processes. Here, we present the X-ray crystal structure of the human αTAT1/acetyl-CoA complex. Together with structure-based mutagenesis, enzymatic analysis, and functional studies in cells, we elucidate the catalytic mechanism and mode of tubulin-specific acetylation. We find that αTAT1 has an overall fold similar to the Gcn5 histone acetyltransferase but contains a relatively wide substrate binding groove and unique structural elements that play important roles in α-tubulin-specific acetylation. Conserved aspartic acid and cysteine residues play important catalytic roles through a ternary complex mechanism. αTAT1 mutations have analogous effects on tubulin acetylation in vitro and in cells, demonstrating that it is the central determining factor of α-tubulin K40 acetylation levels in vivo. Together, these studies provide general insights into distinguishing features between histone and tubulin acetyltransferases, and they have specific implications for understanding the molecular basis of tubulin acetylation and for developing small molecule modulators of microtubule acetylation for therapy.

Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.