MEC-17 is an alpha-tubulin acetyltransferase

Abstract

In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the alpha-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for alpha-tubulin. In vitro, MEC-17 exclusively acetylates K40 of alpha-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R alpha-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 alpha-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of alpha-tubulin, the only PTM known to occur on the luminal surface of microtubules.

Figure 1. MEC-17 is required for acetylation of K40 on α-tubulin in Tetrahymena

a–c, Wild-type (prefed with ink) and MEC17-KO (arrow) cells labeled with anti-acetyl-K40 mAb (6–11 B-1) and anti-tubulin antibodies. d–f, Wild-type (d), MEC17-KO (e) and K40R (f) Tetrahymena labeled with pan anti-acetyl-K antibodies. g–h, Western blots of cells (g) or cytoskeletons (h) probed with 6–11 B-1 mAb, pan anti-acetyl-K, anti-α-tubulin (12G10 mAb) and anti-histone hv1 antibodies. Stars mark non-tubulin proteins. Arrows mark acetylated histones. i, Growth curves of Tetrahymena. j–l, Wild-type (left) and GFP-Mec17p overproducing (right) Tetrahymena cells analyzed for GFP (j) or 6–11 B-1 mAb immunofluorescence (k).

Figure 2. MEC-17 and W06B11.1 are required for acetylation of K40 and contribute to touch sensation in C. elegans

a–f, Wild-type and mutant adult hermaphrodites were labeled using 6–11 B-1 mAb. Small and large arrows mark axons and cell bodies of TRNs, respectively. Scale bar 10 µm. g, Histogram quantifying touch responses. The error bars represent SEM. Asterisk marks significant difference when compared to K40 transgene mec-12(e1607) (p<0.0001). The following numbers of animals were tested: wild type, 69; mec-12(e1607), 49; mec-17(ok2109), 44; W06B11.1(ok2415), 33; mec-17(ok2109) W06B11.1(ok2415), 140; K40 transgene mec-12(e1607), 84; Q40 transgene mec-12(e1607) 78; R40 transgene mec12(e1607) 75.

Figure 3. MEC-17 is required for K40 acetylation in zebrafish and normal embryonic development

Control embryos (a,c,c’,c”,e) and embryos injected with MEC17-ATG morpholinos, 48 hr post fertilization (hpf). (b,d,d’,d”,f) were observed live (a,b) or subjected to immunofluorescence 48 hpf using either 6–11 B-1 mAb (c–d”) or Znp1 mAb (e,f), which recognizes synaptotagmin 1. c’ and d’ show higher magnifications of the areas boxed in c and d. c” and d” show higher magnifications of the areas of pronephrons that contain cilia (marked with arrows in c” and d”). In e and f, arrows mark axons of peripheral neurons.

Figure 4. MEC-17 controls the levels of microtubule acetylation in mammalian cells

a–h, Expression of Mm-MEC-17 in Ptk2 cells increases the levels of acetyl-K40 α-tubulin. Cells expressing either EGFP or EGFP and Mm-MEC17 were stained with 6–11 B-1 mAb and anti-α-tubulin antibodies. i, Depletion of Hs-MEC-17 in HeLa cells reduces the level of acetyl-K40 α-tubulin. Cells were transfected with either GFP or Hs-MEC17 siRNAs and after 50 hr, treated for 7 hr with either 300 nM trichostatin A (TSA, stock solution in DMSO) or DMSO alone. Cell lysates were analyzed by western blot probed with either 6–11 B-1 mAb (top, middle panels) or anti-α-tubulin mAb (bottom panel).

Figure 5. MEC-17 has intrinsic, K40-specific α-TAT activity

a, Crude Tetrahymena and recombinant murine MEC-17 were used for in vitro acetylation reactions of MEC17-KO axonemes and analyzed by western using 6–11 B-1 and 12G10 mAb. b, In vitro acetylation assays were performed with GST-MmMEC-17 using axonemes isolated from either the MEC17-KO (K40) strain or a K40R α-tubulin mutant. The marker (M) is acetylated glutamate dehydrogenase (55.6 kD). c, Recombinant GST-MmMEC-17 directly acetylates purified tubulin from the MEC17-KO strain in vitro. d, Coomassie Blue-stained gel with either purified MEC17-KO tubulin (36 ng) or porcine brain tubulin (15 ng, 99% pure, Cytoskeleton Inc).