The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation

Abstract

Long-lived microtubules found in ciliary axonemes, neuronal processes, and migrating cells are marked by α-tubulin acetylation on lysine 40, a modification that takes place inside the microtubule lumen. The physiological importance of microtubule acetylation remains elusive. Here, we identify a BBSome-associated protein that we name αTAT1, with a highly specific α-tubulin K40 acetyltransferase activity and a catalytic preference for microtubules over free tubulin. In mammalian cells, the catalytic activity of αTAT1 is necessary and sufficient for α-tubulin K40 acetylation. Remarkably, αTAT1 is universally and exclusively conserved in ciliated organisms, and is required for the acetylation of axonemal microtubules and for the normal kinetics of primary cilium assembly. In Caenorhabditis elegans, microtubule acetylation is most prominent in touch receptor neurons (TRNs) and MEC-17, a homolog of αTAT1, and its paralog αTAT-2 are required for α-tubulin acetylation and for two distinct types of touch sensation. Furthermore, in animals lacking MEC-17, αTAT-2, and the sole C. elegans K40α-tubulin MEC-12, touch sensation can be restored by expression of an acetyl-mimic MEC-12[K40Q]. We conclude that αTAT1 is the major and possibly the sole α-tubulin K40 acetyltransferase in mammals and nematodes, and that tubulin acetylation plays a conserved role in several microtubule-based processes.

Fig. 1.

αTAT1 acetylates tubulin through its GNAT domain in vitro. (A) C6orf134/αTAT1 directly binds to the BBSome. GST-αTAT1 and GST were immobilized on beads and mixed with pure BBSome, and bound fractions were immunoblotted for the BBSome subunit BBS4. All panels are from the same film. (B) Recombinant αTAT1 efficiently transfers acetyl groups onto K40 of α-tubulin. αTAT1 (1 μM) was mixed with tubulin and [¹⁴C]acetylCoA and incubated at 24 °C for the indicated times. Reactions were conducted in triplicate and immunoblotted for α-tubulin K40Ac (Top), subjected to phosphorimaging (Middle), Coomassie staining (Bottom) or filtered to count tubulin-bound radioactivity (plotted in C). Coomassie-stained gel shows α/β-tubulin (thick band) and αTAT1 (thin band). (C and D) Time course of acTubulin and αK40Ac formation. Filter-bound radioactivity representing acetyl groups transferred to tubulin was counted and used to calculate the absolute amount of acetylated tubulin (C). At the final time point, the acetylation level reached 0.51 ± 0.02 mol acetyl per mol tubulin. Immunoblot signals were quantified and normalized to the signal intensity at time 0 (D). The level of αK40Ac is increased 12.6-fold at end of reaction. Because ∼5% of bovine brain α-tubulin is acetylated at K40 (42), we calculate that 0.58 ± 0.13 mol of acetyl are transferred to K40 per mol tubulin after 12 h. Error bars are SD and are smaller than some symbols. (E) Purified αTAT1 variants. A 2-μg quantity of each αTAT1 variant was resolved by SDS/PAGE and the gel stained with Coomassie. αTAT1[10-236] is likely to be partially unfolded and contaminated with chaperones. (F) Activity of αTAT1 variants. 1 μM (hatched bars) or 5 μM (solid bars) of each αTAT1 variants were incubated with tubulin and [³H]acetylCoA for 1 h at 24 °C and acetyl incorporation into tubulin measured by filter assay. (G) Summary diagram of truncation study, structural predictions (13), and sequence conservation. The minimal αTAT domain extends N-terminally from predicted GNAT fold, whereas the unstructured and poorly conserved C terminus of αTAT1 enhances catalytic efficiency. (H) Aspartate D157 is crucial for αTAT1 enzymatic activity. Either 5 or 2.5 μM αTAT1[2-236] or αTAT1[2-236]^D157N was incubated with tubulin as in B. No radiolabel incorporation was detected with αTAT1[2-236]^D157N.

Fig. 2.

αTAT1 specifically acetylates K40 of α-tubulin and prefers microtubules over free tubulin. (A) K40 of α-tubulin is the sole site of acetylation by αTAT1. 5 μM αTAT1[2-236] was incubated for 1 h at 37 °C with 4 μM axonemal tubulin purified from either WT or α-tubulin[K40R] Tetrahymena strains and samples processed as in Fig. 1B. Because of high levels of αK40Ac in axonemal microtubules, radiolabel incorporation is less efficient than when using bovine brain tubulin as a substrate. However, no radiolabel incorporation was found after a 3-mo exposure to phosphor screen when α-tubulin[K40R] was used as a substrate. (B) αTAT1 exhibits strict substrate specificity for tubulin vs. histones. Reaction mixtures contained [¹⁴C]acetylCoA and various combinations of αTAT1[2-236] (4 μM), tubulin, HAT1/RbAp46, and histones H3/H4 and were incubated at 37 °C for 1 h. Even in the presence of very high concentrations of αTAT1 enzyme, histone H3/H4 did not serve as an acetylation substrate, whereas HAT1/RbAp46 acetylated histone H4 under the same experimental conditions. (C) αTAT1 displayed a greater catalytic efficiency for taxol-stabilized microtubules than for free tubulin. A range of substrate concentrations straddling the K_m value were incubated for 30 min at 22 °C with 1 μM αTAT1, [³H]acetylCoA, and GTP (microtubules) or GDP (tubulin) and radiolabel incorporation measured with the filter assay. Time-course experiments indicated that the reaction proceeds linearly for the first hour (Fig. 1C). Data were fitted to the Michaelis–Menten equation by nonlinear least-square regression. Tubulin was fully polymerized in the microtubule sample and fully depolymerized in the free tubulin sample, as determined in spindown assays (Fig. S1B). A larger version of the tubulin saturation curve is shown in Fig. S1C. Reactions performed in triplicate. Error bars represent SD and are smaller than some symbols.

Fig. 3.

The enzymatic activity of αTAT1 is necessary and sufficient for acetylation of α-tubulin at lysine 40 in mammalian cells. (A and B) The enzymatic activity of αTAT1 is sufficient for acetylation of α-tubulin at lysine 40 in PtK2 cells. (A) Cells were transfected with GFP-tagged WT αTAT1 (Left), αTAT1[D157N] (Middle), and ELP3 (Right) and stained for DNA (blue), α-tubulin (red), and α-tubulin K40Ac (green). GFP fluorescence was visualized directly. (B) Lysates from transfected cells were immunoblotted for the indicated proteins. All GFP fusions are expressed at similar levels. (C–H) αTAT1 is required for α-tubulin K40 acetylation in RPE-hTERT cells. (E) RPE-hTERT cells were transfected with two independent siRNA duplexes targeting αTAT1 or control siRNA duplexes and stained for the indicated antigens (white) and DNA (blue). (D) Lysates from transfected cells were immunoblotted for the indicated antigens. (C) αTAT1 mRNA levels were measured by RT-qPCR, and values were normalized to the levels in control siRNA-treated cells. GAPDH was used for intrasample normalization purposes. (F) Levels of α-tubulin acetylation at K40 were measured by fluorescent immunoblotting as the ratios of signals for α-tubulin K40Ac over α-tubulin. (G) Growth curve was created by measuring the number of cells in each condition starting 48 h after siRNA transfection. (H) Amount of polymerized tubulin was measured as fraction of pelletable tubulin in lysates. Error bars correspond to SDs of three independent experiments. (Scale bars, 10 μm.)

Fig. 4.

αTAT1 promotes rapid assembly of the primary cilium. (A–C) αTAT1 is required for the normal kinetics of primary cilium assembly. (A) Following siRNA transfection, cells were serum starved, and polyglutamylated tubulin-positive cilia were counted immediately or after 12 or 24 h. Cilia counts were confirmed by examination of the Arl13b channel. Although the difference in cilia number between control and αTAT1-depleted cells is significant at 12 h postserum starvation (P < 0.0001), no significant difference could be detected after 24 h (P > 0.05). Error bars represent SDs among three independent experiments. (B) Cells fixed 12 h after serum starvation were stained for polyglutamylated tubulin (red), Arl13b (green), and DNA (blue). (C) Cells fixed 24 h after serum starvation were stained for polyGlu tubulin (red), α-tubulin K40Ac (green), and DNA (blue). A large proportion of cells are ciliated in the αTAT1-depleted cells. However, α-tubulin K40Ac staining in the cilia of αTAT1-depleted cells is extremely faint. (D and E) Phylogenetic distribution of αTAT1 compared with IFT-A, IFT-B, BBSome, and Arl6. (D) Cilia:molecule correlation coefficients were calculated from Dataset S1 as 100% for αTAT1, 70–98% for IFT-A subunits, 60–96% for IFT-B subunits, and 82–92% for BBSome subunits and Arl6. (E) Simplified taxonomic tree, modified from Carvalho-Santos et al. (43). The full name of each organism is shown in Fig. S4. When present in the organism, the basal body structure is diagramed in black, and the cilium is shown in green (motile) or green/blue (motile and primary) or blue (primary). When no cilium is present, hatched bars fill the row. Membrane is omitted for the Plasmodium cilium to represent IFT-independent flagellum assembly. Presence of αTAT1 ortholog is indicated by a black circle. Conservation of IFT-A and IFT-B complexes and BBSome and Arl6 are depicted with shades of gray that correspond to the proportion of subunits present in the organism (black, 100%; dark gray, <100%; medium gray, <60%; and light gray, <30%).

Fig. 5.

C. elegans mechanosensation requires K40 α-tubulin acetylation. (A) Pmec-17::GFP is expressed only in TRNs (Upper), whereas Patat-2::mCherry is expressed in TRNs, OLQ, CEP, PVR, and neurons in the ventral nerve cord (Lower). (B and C) Both mec-17 and atat-2 are required for α-tubulin K40 acetylation in C. elegans. α-Tubulin K40 acetylation is absent in mec-17;atat-2 double null mutants and mec-12 α-tubulin null mutants. Bars are normalized to WT intensity (n = 3). (D and E) α-tubulin K40Ac is present in OLQ, CEP, and ALM in the head (D) and PVR and PLM in the tail (E). For clarity, larger micrographs are shown in. Fig. S5. (F) Loss of αTAT-2, but not MEC-17 decreases sensitivity to nose touch (n = 30 worms/genotype). *P < 0.01 vs. WT, one-way ANOVA F(4,145) = 154.3, P = 10⁻⁵¹, Tukey post hoc test. (G) Loss of both αTAT proteins decreases, but does not eliminate, responses to body touch (n = 75 worms per genotype). *P < 0.01 vs. WT, one-way ANOVA F(4,370) = 246.5, P = 10⁻¹⁰³, Tukey post hoc test. (H) Transgenic expression of WT, but not catalytically inactive MEC-17 or MEC-12[K40R], restores touch sensitivity to mec-17 and mec-12 null mutants, respectively (Left and Center). Transgenic expression of acetyl-mimic MEC-12[K40Q] restores touch sensitivity to mec-17;atat-2;mec-12 triple null mutants (Right) (n = 75 worms per genotype). *P < 0.01, Student t test. Bars in all panels represent mean ± SEM.