Dorota Wloga

MEC-17 is an α-tubulin acetyltransferase

In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the ε-amino group of K40 on α-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 α-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 α-tubulin. In vitro, MEC-17 exclusively acetylates K40 of α-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R α-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 α-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 α-tubulin, the only PTM known to occur on the luminal surface of microtubules.

Post-translational modifications of microtubules

Microtubules – polymers of tubulin – perform essential functions, including regulation of cell shape, intracellular transport and cell motility. How microtubules are adapted to perform multiple diverse functions is not well understood. Post-translational modifications of tubulin subunits diversify the outer and luminal surfaces of microtubules and provide a potential mechanism for their functional specialization. Recent identification of a number of tubulin-modifying and -demodifying enzymes has revealed key roles of tubulin modifications in the regulation of motors and factors that affect the organization and dynamics of microtubules.

TTLL3 Is a Tubulin Glycine Ligase that Regulates the Assembly of Cilia

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffers vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

Evolutionary Divergence of Enzymatic Mechanisms for Posttranslational Polyglycylation

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this posttranslational modification, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.

Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila

ABSTRACT Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development

BackgroundThe post-translational addition of the monosaccharide O-linked β-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.ResultsWe manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish ogt, human oga or by injecting morpholinos against ogt transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor spiel ohne grenzen. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells.ConclusionWe conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.

Chapter 4 Ciliary Tubulin and Its Post-Translational Modifications

Tubulin, the most abundant axonemal protein, is extensively modified by several highly conserved post-translational mechanisms including acetylation, detyrosination, glutamylation, and glycylation. We discuss the pathways that contribute to the assembly and maintenance of axonemal microtubules, with emphasis on the potential functions of post-translational modifications that affect tubulin. The recent identification of a number of tubulin modifying enzymes and mutational studies of modification sites on tubulin have allowed for significant functional insights. Polymeric modifications of tubulin (glutamylation and glycylation) have emerged as important determinants of the 9 + 2 axoneme assembly and motility.

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

ABSTRACT In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.

Septins Stabilize Mitochondria in Tetrahymena thermophila

ABSTRACT We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.