Molecular Basis for Age-Dependent Microtubule Acetylation by Tubulin Acetyltransferase

Abstract

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule inter-protofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate-limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

Mechanism of microtubule lumen entry for the α-tubulin acetyltransferase enzyme αTAT1

“…4D). In addition, we found that αTAT1 does not acetylate tubulin dimers as efficiently as it does intact microtubules, consistent with previous αTAT1 studies (18,19) (Fig. 4E, blue).…”

“…However, despite identification of the enzyme and its substrate, the mechanism for how αTAT1 enters the microtubule lumen to access its acetylation sites is yet to be fully understood. There are several potential mechanisms for how αTAT1 may access the acetylation site on the inside of the microtubule lumen: by copolymerization with tubulin at growing microtubule plus-ends (15), by transient openings in the lattice during microtubule breathing (13,16,17), or by microtubule end-entry (12,18). Copolymerization is not likely to be the primary mechanism for αTAT1 access, because stable microtubules are acetylated, and because αTAT1 is more active on polymerized microtubules than on free tubulin dimers (12,13,(19)(20)(21)(22)(23)(24)(25).…”

“…Copolymerization is not likely to be the primary mechanism for αTAT1 access, because stable microtubules are acetylated, and because αTAT1 is more active on polymerized microtubules than on free tubulin dimers (12,13,(19)(20)(21)(22)(23)(24)(25). Conversely, the other modes of access are possible (12,13,18). Distinguishing among these possibilities is important for developing a mechanistic understanding to explain how αTAT1 could access its acetylation sites to facilitate microtubule acetylation.…”

“…Further, recent work suggested that αTAT1 was able to diffuse efficiently within the microtubule lumen, and thus acetylate microtubules without a preference for microtubule ends (18). However, our results support a model in which the mobility of αTAT1 within the lumen is controlled by the affinity of αTAT1 for its acetylation sites, which are highly concentrated inside of the microtubule lumen, and in which the acetylation efficiency of αTAT1 is regulated, in part, by the accessibility of acetylation sites through alterations in microtubule structure.…”

Methamphetamine promotes α-tubulin deacetylation in endothelial cells: The protective role of acetyl-l-carnitine

“…Cytoskeleton microtubules are regulated by several types of conserved posttranslational modifications (Song and Brady, 2014). Although the biological meaning of such mechanisms is still poorly understood, it was reported that tubulin acetylation increases its stability and half-life, rendering microtubules more resistant to druginduced depolymerization and disassembly (Matsuyama et al, 2002;Szyk et al, 2014). Previous studies showed that METH impacts actin filaments, leading to actin depolymerization, altered cell shape and local accumulation of condensed actin, which may compromise TJs function and BBB integrity (Park et al, 2013;Young et al, 2014).…”