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The Major Α-Tubulin K40 Acetyltransferase Αtat1 SEE COMMENTARY Promotes Rapid Ciliogenesis and Efficient Mechanosensation

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The Major Α-Tubulin K40 Acetyltransferase Αtat1 SEE COMMENTARY Promotes Rapid Ciliogenesis and Efficient Mechanosensation The major α-tubulin K40 acetyltransferase αTAT1 SEE COMMENTARY promotes rapid ciliogenesis and efficient mechanosensation Toshinobu Shida, Juan G. Cueva, Zhenjie Xu, Miriam B. Goodman1, and Maxence V. Nachury1 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5345 Edited* by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved October 20, 2010 (received for review September 15, 2010) Long-lived microtubules found in ciliary axonemes, neuronal pro- uncharacterized protein, together with the BBSome (12), and we cesses, and migrating cells are marked by α-tubulin acetylation on confirmed that C6orf134 directly binds to the BBSome (Fig. 1A). lysine 40, a modification that takes place inside the microtubule Intriguingly, C6orf134 is predicted to harbor the GNAT fold lumen. The physiological importance of microtubule acetylation found in HATs (13). We thus hypothesized that C6orf134 is the remains elusive. Here, we identify a BBSome-associated protein long-sought TAT responsible for K40 acetylation of α-tubulin. that we name αTAT1, with a highly specific α-tubulin K40 acetyl- To test this assumption, we assayed recombinant C6orf134 for transferase activity and a catalytic preference for microtubules enzymatic transfer of acetyl from acetyl-CoA onto tubulin. over free tubulin. In mammalian cells, the catalytic activity of Consistent with our hypothesis, we found robust incorporation of αTAT1 is necessary and sufficient for α-tubulin K40 acetylation. radiolabeled acetyl into tubulin—as much as 0.51 mol/mol (Fig. Remarkably, αTAT1 is universally and exclusively conserved in cil- 1C)—and a concomitant increase in the immunoreactivity for iated organisms, and is required for the acetylation of axonemal mAb 6-11B-1 (Fig. 1 B and D), an antibody that specifically 14 microtubules and for the normal kinetics of primary cilium assem- recognizes α-tubulin K40Ac (14). The time courses of [ C]acetyl bly. In Caenorhabditis elegans, microtubule acetylation is most incorporation and K40Ac synthesis were similar (Fig. 1 C and prominent in touch receptor neurons (TRNs) and MEC-17, a homo- D). Similar findings were reported independently (15). C6orf134 log of αTAT1, and its paralog αTAT-2 are required for α-tubulin was thus renamed αTAT1. acetylation and for two distinct types of touch sensation. Further- The boundaries of the predicted GNAT fold and of the minimal more, in animals lacking MEC-17, αTAT-2, and the sole C. elegans acetyltransferase domain of αTAT1 are nearly identical (Fig. 1 E– K40α-tubulin MEC-12, touch sensation can be restored by expres- G), suggesting that the mechanism of acetyl group transfer is sion of an acetyl-mimic MEC-12[K40Q]. We conclude that αTAT1 is conserved between αTAT1 and HATs. To test this idea and to the major and possibly the sole α-tubulin K40 acetyltransferase in generate a catalytically inactive αTAT1, we searched for conserved mammals and nematodes, and that tubulin acetylation plays a con- acidic residues, as most GNAT family members use an acidic res- served role in several microtubule-based processes. idue for catalysis. We identified D157 and found that αTAT1 [D157N] failed to acetylate purified tubulin (Figs. 1 F and H). Thus, α cytoskeleton | posttranslational modification | cilia | the TAT activity resides within TAT1 and not a contaminant. Bardet-Biedl Syndrome | ciliopathy Recent proteome-wide studies have found multiple acetylated lysines on α- and β-tubulin (16). We therefore tested whether lysines other than α-tubulin K40 are acetylated by αTAT1 in vitro. ong-lived, stable microtubules are found inside neuronal Although αTAT1 acetylates tubulin purified from WT Tetrahy- processes and cilia and provide stable tracks for motors. L mena, it is unable to acetylate tubulin purified from an α-tubulin Stable microtubules also play essential and conserved roles in the [K40R] knock-in Tetrahymena strain (Fig. 2A). Thus, αTAT1 sense of touch and harbor a variety of posttranslational mod- fi α ifications such as detyrosination, polyglutamylation, and N- speci cally acetylates K40 of -tubulin. In further support of this ε fi conclusion, we calculated that the total amount of acetyl deposited acetylation of -lysines (1). Although most known modi cations fi are displayed on the outside of the microtubule lattice, the major onto tubulin is nearly identical to the amount of acetyl speci cally incorporated at K40 of α-tubulin (Fig. 1 C and D). acetylation site in ciliary microtubules is located on lysine-40 of fi α-tubulin, a residue that points into the lumen of microtubules All protein lysine acetyltransferases (KAT) identi ed to date (2). Thus far, the functional consequences of α-tubulin K40 have been HATs, and some HATs acetylate nonhistone substrates fi α acetylation have remained perplexing, as genetic alteration of (17). To determine the substrate speci city of TAT1, we in- α α-tubulin K40 has failed to produce detectable phenotypes in cubated recombinant TAT1 with either recombinant histone H3/ either protozoa (3, 4) or nematodes (5). Nonetheless, recent H4 (Fig. 2B) or purified core histones (Fig. S1A) and found that evidence suggest that kinesin-1 preferentially travels on acety- αTAT1 is unable to detectably acetylate any of the core histones, lated microtubule tracks (6, 7) and that α-tubulin K40 acetylation whereas HAT1/RbAp46 efficiently acetylates histone H4 under is required for radial glial cell migration (8). These various data the same conditions. Furthermore, HAT1/RbAp46 was unable to have made the physiological role of α-tubulin K40 acetylation acetylate tubulin (Fig. S1A). Thus, αTAT1 is a KAT that is not subject to debates that have not been resolved because of a lack a HAT. of tools for eliminating α-tubulin K40 acetylation. In particular, although two α-tubulin K40 deacetylases are known, the identity of the α-tubulin K40 acetyltransferase (αK40 TAT or αTAT) Author contributions: T.S., J.G.C., Z.X., M.B.G., and M.V.N. designed research; T.S., J.G.C., CELL BIOLOGY remains unknown (1), despite recent suggestions that some his- Z.X., and M.V.N. performed research; T.S., J.G.C., Z.X., M.B.G., and M.V.N. analyzed data; tone acetyltransferases (HATs) and N-acetyltransferases in- and M.B.G. and M.V.N. wrote the paper. fluence microtubule acetylation (9, 10). The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. Results See Commentary on page 21238. fi A BBSome-Associated Protein Speci cally Acetylates Lysine 40 of 1To whom correspondence may be addressed. E-mail: [email protected] or α-Tubulin in Vitro. We previously showed that a subunit of the [email protected]. BBSome, a ciliary trafficking complex, is required for α-tubulin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. K40 acetylation (11). Recently, we copurified C6orf134, an 1073/pnas.1013728107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1013728107 PNAS | December 14, 2010 | vol. 107 | no. 50 | 21517–21522 Downloaded by guest on September 26, 2021 A E A WT K40R WT K40R C D157N Microtubules Input αTAT1 GST MW K = 1.6 ± 0.36 μM BBS4 14 m (kDa) [10-236][2-193][2-207][2-236][2-236]FL Coomassie [ C] k = 615 ± 34 x 10-6 s-1 B cat bound 116 αTAT1 ++ ++ 97 60 time (h) HAT1/RbAp46 + + B 66 Tubulin + + 50 01246912 M/s) Histone H3/H4 + + + +++ MW (kDa) μ α-tubulin 40 45 HAT1/ 66 -5 K40Ac RbAp46 30 Free Tubulin [14C] 31 Tubulin (x 10 45 i μ Km= 2.0 ± 0.16 M 21 V 20 α k = 98 ± 1.8 x 10-6 s-1 Coomassie 14 TAT1 cat 31 10 F [αTAT1] 21 7 M) H3 C 5 μM 0 μ [10-236] H4 14 0 5 10 15 20 25 6 μ 6 μ [2-193] 1 M 14 [Tubulin] ( M) 5 Coomassie [ C] [2-207] 4 [2-236] Fig. 2. αTAT1 specifically acetylates K40 of α-tubulin and prefers micro- 3 [2-236]D157N tubules over free tubulin. (A) K40 of α-tubulin is the sole site of acetylation 2 FL by αTAT1. 5 μM αTAT1[2-236] was incubated for 1 h at 37 °C with 4 μMax- 1 0 5 10 15 20 onemal tubulin purified from either WT or α-tubulin[K40R] Tetrahymena 0 strains and samples processed as in Fig. 1B. Because of high levels of αK40Ac [acTubulin] formed ( 024681012 % mol ac/mol tub fi time (h) G in axonemal microtubules, radiolabel incorporation is less ef cient than 47 conserved 190 when using bovine brain tubulin as a substrate. However, no radiolabel in- D 14 2 193 333 α corporation was found after a 3-mo exposure to phosphor screen when minimal TAT domain unstructured α α 12 15 GNAT fold 189 -tubulin[K40R] was used as a substrate. (B) TAT1 exhibits strict substrate specificity for tubulin vs. histones. Reaction mixtures contained [14C]ace- 10 α α TAT1 TAT1 tylCoA and various combinations of αTAT1[2-236] (4 μM), tubulin, HAT1/ 8 H WT D157N WT D157N RbAp46, and histones H3/H4 and were incubated at 37 °C for 1 h. Even in the 6 presence of very high concentrations of αTAT1 enzyme, histone H3/H4 did Tubulin 4 not serve as an acetylation substrate, whereas HAT1/RbAp46 acetylated 2 histone H4 under the same experimental conditions. (C) αTAT1 displayed 1 αTAT1 -tubulin K40Ac level (RU) K40Ac level -tubulin 024681012 a greater catalytic efficiency for taxol-stabilized microtubules than for free 14 α time (h) [ C] Coomassie tubulin. A range of substrate concentrations straddling the Km value were incubated for 30 min at 22 °C with 1 μM αTAT1, [3H]acetylCoA, and GTP Fig.
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