Jpn. J. Protozool. Vol. 43, No. 1. (2010) 5

Review

Tubulin polymodifications in Tetrahymena

Jacek GAERTIG

Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA

Ciliates have a -rich cytoskeleton. isotypes in Tetrahymena Tetrahymena thermophila assembles at least 18 types of distinct that are located in Early studies suggested that T. thermophila con- cilia, cell cortex, nuclei and cytoplasm of the cell tains only single isotypes of D- and E-tubulin body. Specific microtubules differ in the filament (Gaertig et al., 1993; McGrath et al., 1994). This length and curvature, degree of bundling, level of view has been revised on the basis of the sequence subunit turnover and levels of microtubule- of the macronuclear genome (Eisen et al., 2006). interacting proteins. How diverse microtubules Tetrahymena has only a single gene, ATU1, encod- assemble and function is a largely unanswered ing a conventional D-tubulin, Atu1p (McGrath et question, to which ciliate studies have recently al., 1994) and two genes for a conventional E- offered important insights. Commonly, multiple tubulin (BTU1 and BTU2) that encode exactly the variants of the microtubule building blocks, same protein, Btu1/2p (Gaertig et al., 1993). The dimers of D-/E-tubulin, are expressed in the same sequences of Atu1p and Btu1/2p are cell. Often tubulin variants are spatially segre- over 90% identical to orthologs in most eukaryo- gated, and therefore could be involved in the tes. Not surprisingly, Tetrahymena needs conven- functional adaptations of specific microtubules. tional D- and E-tubulin for survival. ATU1 is es- Tubulin variants arise by: 1) expression of dis- sential (Hai et al., 1999). While the expression tinct tubulin isotypes (products of distinct genes) patterns of BTU1 and BTU2 are distinct (Gu et al., and 2) production of tubulin isoforms by post- 1995), Tetrahymena needs only one of them for translational modifications. survival (Xia et al., 2000). In addition, Tetrahy- mena has single genes encoding highly conserved that are associated with the sites of micro- *Corresponding author tubule nucleation, including J- G-, K- and H-tubulin Tel: +1 706 542 3409 (Eisen et al., 2006; Shang et al., 2002). The J- Fax: +1 706 542 4271 tubulin of Tetrahymena is essential and required @ . . E-mail: jgaertig cb uga edu for duplication and maintenance of basal bodies Received: 13 August 2009 (Shang et al., 2002). The G, K- and H-tubulin have

6 原生動物学雑誌 第43巻 第1号 2010年 not been studied in Tetrahymena. However, these McKanna, 1973). This is a rare example of micro- tubulins are essential in Paramecium. G-tubulin, as tubules with an extreme angle of curvature. The shown earlier in Chlamydomonas (Dutcher and CVP microtubules may require an unusual degree Trabuco, 1998), is required for the assembly of the of flexibility, which could involve unusual bonds C-tubule in triplet microtubules of the basal body between tubulin subunits. In the future, it will be (Garreau De Loubresse et al., 2001). The K- and H- of interest to determine whether divergent tubulins tubulin are required for duplication of basal bodies of ciliates have amino acid substitutions at do- (Dupuis-Williams et al., 2002; Ruiz et al., 2000). mains that are known to form inter- and intra- Thus ciliates have a set of conserved conventional dimer surfaces and thus affect polymer flexibility tubulins, most if not all of which are essential. (Nogales et al., 1999; Nogales et al., 1998). However, the genome of Tetrahymena also con- tains predicted genes encoding 3 extremely diver- gent D-tubulin-like proteins (ALT1 to 3) and 9 Tubulin isoforms in Tetrahymena equally divergent E-tubulin-like proteins (BLT1 to 6, and 3 so called L-tubulins) that appear to be cili- Tubulin of most eukaryotes undergoes a number ate-specific (Aury et al., 2006; Eisen et al., 2006). of conserved post-translational modifications Due to their similarity to D- and E-tubulins, the D- (PTMs). Tetrahymena tubulin is highly heteroge- like and E-like tubulins could be used as building neous and extensively modified post- blocks of microtubules. Thus, contrary to the ear- translationally (Gaertig et al., 1995; Redeker et al., lier assumptions (Gaertig et al., 1993; McGrath et 2005; Suprenant et al., 1985). The most conserved al., 1994), isotypic diversity could play a major and well studied tubulin PTMs that are also pre- role in ciliates. In vegetative cells, Atu1p and sent in ciliates (Fig. 1) include: of D- Btu1/2p, are incorporated into all types of micro- tubulin (at K40) (Greer et al., 1985), proteolytic tubules (Thazhath et al., 2004 and our unpublished removal of the terminal Y residue from D-tubulin data). It is likely that the divergent tubulins (D- () (Argarana et al., 1978), and two like, E-like and L-) do not form separate micro- types of so-called polymodifications, glutamyla- tubules, but instead co-assemble with conventional tion (Eddé et al., 1990) and glycylation (Redeker tubulin. Since the divergent tubulin isotypes may et al., 1994) (on D- and E-tubulin). With exception be specific to ciliates, these proteins could be in- of acetylation at K40 of D-tubulin that occurs in- volved in assembly of types of microtubules that side the microtubule lumen (Nogales et al., 1999), are unique to ciliates. Indeed, in Tetrahymena, an the remaining PTMs occur on the outside surface epitope tagged Blt1p, is not targeted to axonemes of microtubules. More precisely, detyrosination, (K. Clark, M. Gorovsky, personal communica- glutamylation and glycylation occur on the C- tion), organelles that contain mostly if not entirely terminal tails (CTTs) of tubulin, flexible domains conventional tubulin dimers (Gaertig et al., 1995). (Luchko et al., 2008) that interact with motor pro- The divergent D- and E-tubulins could co-assemble teins and other types of microtubule-associated with conventional tubulin in microtubules with proteins (MAPs) (Roll-Mecak and Vale, 2008; unusual properties such as the cortical bundles, Skiniotis et al., 2004; Wang and Sheetz, 2000). microtubules that form during amitosis of the mac- Tetrahymena studies showed that the CTT on both ronucleus (Fujiu and Numata, 2000) or those form- D- and E-tubulin is essential (Duan and Gorovsky, ing the contractile vacuole pore (CVP). CVP con- 2002). tains a small ring of microtubules twisted into a Here we will focus on PTMs that are based on right-handed helix (Elliott and Bak, 1964; ligations of amino acids to tubulin, and in particu- Jpn. J. Protozool. Vol. 43, No. 1. (2010) 7

Fig. 1. Immunofluorescence images of a dividing Tetrahymena labeled with antibodies that recognize either the tubulin primary sequence (A) or various post-translational form of tubulin. The following antibodies were used: (B) GT335, a monoclonal antibody (mAb) that recognizes mono- and polyglutamyl side chain (Wolff et al., 1992), (C) ID5, a mAb that in Tetrahymena is specific to (Rüdiger et al., 1999; Wloga et al., 2008), (D) 6-11 B-1, a mAb against acetyl-K40 on D-tubulin (Piperno and Fuller, 1985), (E), TAP952, a mAb against monoglycylated tubulin (Bré et al., 1998) and (F), AXO49, a mAb that recognizes polyglycy- lated tubulin (Bré et al., 1998).

lar on the mechanism and function of polymodifi- mal tubulin of Paramecium is extensively modi- cations (glycylation and glutamylation). Tubulin fied by ligation of (glycylation) (Redeker undergoes post-translational ligations of amino et al., 1994). Unlike tubulin tyrosination that is acids to the primary polypeptide that utilize protein based on a standard , glycylation and translation-independent mechanisms. These reac- glutamylation involve isopeptide bonds that utilize tions are mediated by ATPase enzymes that func- the J-carboxyl group in the side chain of an inter- tion as amino acid ligases (see below). Arce, Arga- nal of CTT. The first added rana and colleagues first reported that D-tubulin is or glutamate can be extended into a side chain by modified by post-translational ligation of standard isopeptide bonds (Redeker et al., 1991; to the C-terminal glutamic acid (Arce et al., 1975; Regnard et al., 1998; Rogowski et al., 2009; Wolff Argarana et al., 1977). Tyrosination is a reverse et al., 1994). To some extent, polymodifications reaction for a post-translational modification based resemble PTMs based on post-translational liga- on proteolytic removal of the genome-encoded C- tion of peptides, (e.g. ubiquitination or neddyla- terminal tyrosine by a carboxypeptidase (Argarana tion), except that the unit of polymodifications is a et al., 1978; Hallak et al., 1977). Eddé and col- single amino acid. Due to the polymeric nature, leagues discovered that murine brain tubulin un- glycylation and glutamylation generate a large dergoes post-translational addition of one or multi- number of tubulin isoforms as a consequence of ple glutamic acids (glutamylation) (Eddé et al., utilization of multiple modification sites (glutamic 1990). Redeker and colleagues showed that axone- acids within the CTTs), variable length of side 8 原生動物学雑誌 第43巻 第1号 2010年 chains and the fact that both polymodifications cific number of E residues. Perhaps the most com- often co-exist on the same tubulin subunits in vari- plete picture of glutamyl side chain distribution ous combinations (reviewed in (Gaertig and emerged from our recent studies in Tetrahymena Wloga, 2008). It came as a surprise, that despite (Wloga et al., 2008). It appears that every type of a major differences between the biochemical mecha- microtubule in Tetrahymena contains at least some nisms of tyrosination and polymodifications, all 3 subunits that have glutamyl side chains. However, PTMs are generated by structurally related en- the upper limit of the glutamyl side chain is micro- zymes (see below). tubule type-specific. Intracytoplasmic, nuclear microtubules and a subset of cortical microtubules have side chains limited to monoglutamylation. Tubulin glutamylation Side chains with an upper limit of 2 Es (biglutamylation) are present in the postoral fiber Tubulin glutamylation is present in most eu- and CVP. Basal bodies and cilia are the only loca- karyotes with a possible exception of fungi. The tions containing side chains composed of 3 or phylogenic patterns suggest that this PTM co- more Es (Wloga et al., 2008). However, in loca- evolves with cilia and centrioles/basal bodies tions that contain the longest side chains there is a (Janke et al., 2005). While, axonemes and centri- whole range of tubulin subunits having from 0 to oles/basal bodies are highly enriched in glutamy- 20 Es per tubulin (Redeker et al., 2005). Moreover, lated tubulin, typically in the same cell, this PTM the distribution of glutamylated subunits within the is also present on other types of microtubules. For microtubule is far from random. Immunofluores- example, mammalian fibroblasts have high levels cence studies in several ciliated models showed of tubulin glutamylation in primary cilia and the that in the axoneme, the density of glutamyl side centrosome, but the PTM is weakly detectable on chains changes, forming an increasing gradient cytoplasmic and spindle microtubules (Bobinnec et from the tip of cilia toward the basal body al., 1998; van Dijk et al., 2007). Bré and col- (Fouquet et al., 1994; Huitorel et al., 2002; Kann et leagues have documented the distribution of gluta- al., 1995; Lechtreck and Geimer, 2000). Strikingly, mylated tubulin in Paramecium and Tetrahymena within the peripheral doublet microtubule, most of by immunofluorescence with a glutamylation- glutamylation is located in the B-tubule specific antibody (Bré et al., 1994) and later con- ((Lechtreck and Geimer, 2000; Multigner et al., firmed by mass spectrometry of purified tubulin of 1996) and our unpublished results for Tetrahy- Tetrahymena (Redeker et al., 2005). In both Para- mena). Thus, enzymes that generate glutamylation mecium and Tetrahymena, glutamylation is present (E-ligases, see below) may be selectively binding in most if not all types of microtubules. However, to specific microtubule surfaces. the extent of glutamylation is spatially regulated. One straightforward mechanism that could con- The formation of the glutamyl side chain consists trol the side chain density and length is the age of of two distinct steps: 1) initiation based on a the microtubule polymer. There are major differ- isopeptide peptide bond that involves the J - ences in the rate of subunit exchange among spe- carboxyl group of the glutamic acid in the primary cific types of microtubules. In Tetrahymena, sequence and 2) elongation based on a standard ³SXOVH-FKDVH´ W\SH H[SHULPHQWV ZLWK DQ HSLWRSH- (Redeker et al., 1991; Regnard et tagged tubulin showed that cytoplasmic, nuclear, al., 1998; Wolff et al., 1994). A number of mono- and longitudinal cortical (LM) microtubules turn- and polyclonal antibodies have been developed over rapidly, while basal body, transverse, post- that recognize glutamyl side chains made of a spe- ciliary and axonemal microtubules turn over very Jpn. J. Protozool. Vol. 43, No. 1. (2010) 9 slowly (Thazhath et al., 2004). The types of micro- microtubules have side chains limited to monogly- tubules that have short glutamyl side chains are cylation (Thazhath et al., 2004; Thazhath et al., mostly the dynamic cell body microtubules that 2002; Xia et al., 2000). Mass spectrometry studies have a short life span. Thus, these microtubules on ciliary tubulin detected from 0-42 G residues may not exist for a sufficient time to acquire long per tubulin (Redeker et al., 2005). However, as is glutamyl side chains. On the other side, most the case of glutamylation, within the organelle, or microtubules that exchange subunits very slowly, possibly a single microtubule, glycylation is dis- such as basal bodies and cilia, also have the long- tributed non-uniformly. Within the axoneme of est glutamyl side chains. While the microtubule Tetrahymena, both mono- and age could play some role, additional mechanisms could not be detected on the central pair micro- are needed for generation the precise pattern of tubules, but were abundant on the peripheral dou- glutamylation, including restrictions to specific blets (Wloga et al., 2009). It is not known yet microtubule surfaces. Furthermore, in Tetrahy- whether, like glutamylation, glycylation is also mena, artificial stabilization of dynamic monoglu- confined to the B-tubule of outer doublets in Tet- tamylated microtubules by paclitaxel does not lead rahymena, but this is the case of axonemes of sea to accumulation of long glutamyl side chains (our urchin sperm (Multigner et al., 1996). Multiple unpublished data). This argues that the length of adjacent glutamic acids in the tail domains of both glutamyl side chains is primarily regulated at the D- and E-tubulin are used for glycylation, based on level of activity of tubulin modifying or demodify- mass spectrometry studies in Paramecium and ing enzymes. mutational studies that located homologous sites in Tetrahymena (Vinh et al., 1999; Xia et al., 2000).

Tubulin glycylation Significance of sites of polymodifications on The first hint of the existence of tubulin glycyla- tubulin tion came from studies that utilized antibodies generated against axonemal tubulin of Parame- Our laboratory has studied the significance of cium tetraurelia. These antibodies recognized E residues on CTTs of D- and E-tubulin in Tetra- ciliary tubulin in a variety of species but often hymena that either are homologous to residues failed to cross-react with non-ciliary cytoplasmic known to serve as sites of polymodifications in microtubules in the same cells (Adoutte et al., other organisms or are adjacent to these sites. 1985). Mass spectrometry showed that these Charge-conserving amino acid substitutions of axoneme-tubulin specific antibodies recognize a CTT (E to D) were used to assess how important polyglycine side chain attached to either D- or E- the polymodification sites are in vivo. These stud- tubulin. While generally, tubulin glycylation is ies have benefited greatly from the use of hetero- found only in cells with cilia (Levilliers et al., karyon strains that have disruptions of ATU1 or 1995), in ciliates, this PTM is also present on non- BTU1 and BTU2 genes in the micronucleus. When ciliary microtubules (Adoutte et al., 1991; Iftode et tubulin heterokaryons mate, their progeny dies but al., 2000; Thazhath et al., 2002). In Tetrahymena could be rescued by introduction of a correspond- the spatial distribution of glycylation resembles ing tubulin gene (Hai et al., 1999; Xia et al., 2000). that of glutamylation. Thus, the long side glycyl The rescue method was used to rapidly assess the chains are present in cilia, basal bodies and cortical significance of multiple sites of polymodifications. microtubules, while cytoplasmic and nuclear Sites of polymodifications on the Atu1p D-tubulin 10 原生動物学雑誌 第43巻 第1号 2010年 are not essential (Wloga et al., 2008; Xia et al., target sites on one tubulin subunit, modify similar 2000). In fact, all 5 glutamic acids of CTT on D- sites on the partner subunit. In fact, this has been tubulin could be replaced by aspartates, leading to observed for some tubulin E-ligases (van Dijk et complete loss of polymodifications from the D- al., 2007). It is not known whether excessive levels tubulin subunit. The resulting mutant cells (ATU1- of polymodifications on the partner subunit in the 5D) are viable but grow more slowly (Wloga et al., tubulin polymodifications site mutants are com- 2008). The tail domain of Btu1/2p E-tubulin con- pensating or contribute to the mutant phenotype. tains five Es that are homologous to the glycylated To determine the consequences of loss-of-function residues in Paramecium (Vinh et al., 1999; Xia et of polymodifications sites, future studies are al., 2000). Tetrahymena can tolerate substitutions needed to mutate the glutamic acids on both D- and of 1 or 2 specific polymodifiable Es; however, E-tubulin in a single strain of Tetrahymena. triple or quadruple mutations are lethal or deleteri- Importantly, in Tetrahymena, the phenotype of a ous (Xia et al., 2000). Mutants with a lethal triple lethal triple mutation in the polymodification do- site mutation develop a novel phenotype: undergo main on E-tubulin is almost precisely phenocopied 3-4 cell cycles without completing cytokinesis and by knockouts of genes that encode subunits of IRUP ³FHOO-FKDLQV´ 7KHVH FRPSRXQG FHOOV FDQ katanin, a microtubule-severing protein (Sharma et assemble only excessively short disorganized cilia al., 2007). These observations open a possibility that lack central microtubules and have defects in that katanin and polymodifications act in the same the peripheral doublet microtubules (Thazhath et pathway. One possibility is that katanin selectively al., 2004; Thazhath et al., 2002). The cytokinesis recognizes tubulin subunits that are polymodified. arrest in the lethal triple polymodification site mu- Katanin and related spastin bind to the tubulin tant is associated with lack of severing of LM cor- CTT (McNally and Vale, 1993; Roll-Mecak and tical bundles (Thazhath et al., 2004; Thazhath et Vale, 2008; White et al., 2007). An antibody that al., 2002). It is likely that the a failure to sever binds to a terminal glutamic acid (to either a dety- LMs blocks the ingression of the cleavage furrow rosinated end of D-tubulin or glutamyl side chain), (Thazhath et al., 2004). These studies indicate that blocks spastin-mediated microtubule severing ac- Es that serve as polymodification sites are essen- tivity in vitro (Roll-Mecak and Vale, 2008). A tial. The limitation of these studies is that these deficiency in katanin activity could lead to lack of sites could potentially be used by both glutamyla- severing of LM microtubules during cytokinesis tion and glycylation and the sites used by glutamy- and this could explain the cytokinesis arrest in the lation have not been mapped for Tetrahymena. triple polymodification site E-tubulin mutants of Moreover, it is not clear to what extent the ob- Tetrahymena. Indeed a GFP-katanin fusion protein served phenotypes of mutations on E-tubulin are localizes to LMs in Tetrahymena (Sharma et al., loss or gain-of-function. For example, substitutions 2007). It is more difficult to explain how katanin of polymodification sites on one of the two tubulin contributes to assembly of cilia. It appears to ka- subunits, change the composition of polymodifica- tanin acts inside cilia, possibly on the outer doublet tions on the partner (non-mutated) subunit. In a microtubules, that are also heavily polymodified viable E-tubulin mutant lacking 3 adjacent poly- (Dymek et al., 2004; Sharma et al., 2007). It re- modification sites, there is an increase in the level mains to be determined whether in the context of of glycylation and decrease in the level of gluta- ciliary assembly, katanin acts as a microtubule mylation on the non-mutated D-tubulin subunit severing factor, or mediates another related activ- (Redeker et al., 2005). It is possible that some ity on the surface of microtubules. modifying enzymes, in the absence of high affinity Jpn. J. Protozool. Vol. 43, No. 1. (2010) 11

The enzymes that generate tubulin polymodifi- served enzymatic properties. It appears that the cations (E- and G-ligases) major differences among the E-ligases of distinct TTLL classes are based on 1) the preference for Į- Regnard and colleagues developed an in vitro or ȕ-tubulin and 2) ability to either initiate or elon- assay for tubulin glutamylation and partially puri- gate the side chains. As an example, TTLL1 pro- fied a tubulin E-ligase activity from murine brains teins (based on studies in Tetrahymena and mouse (Regnard et al., 1998; Regnard et al., 1999). Strik- studies) have an E-ligase activity that prefers Į- ingly, one of the proteins in the complex was tubulin and in Tetrahymena this enzyme has a found to be TTLL1, a protein with a conserved strong side chain initiating activity (Janke, 2005; domain related to a tubulin enzyme previously van Dijk et al., 2007; Wloga et al., 2008). Dele- purified by Weber laboratory, tubulin tyrosine tions of a TTLL1 type enzyme (Ttll1p) and a ligase (Janke et al., 2005). TTL is responsible for closely related TTLL9 type enzyme (Ttll9p) in ligation of tyrosine to the terminal glutamic acid Tetrahymena led to a major loss of glutamylation that is exposed by detyrosination (Schroder et al., in the basal bodies (Wloga et al., 2008). Double 1985). While TTL (Y-ligase) is a reverse PTM knockout cells grow more slowly and have fewer enzyme and E-ligase is a forward PTM enzyme, ciliary rows. Basal bodies with an apparently ma- there are similarities between the reactions cata- ture morphology that failed to dock at the plasma lyzed by these two enzymes. Namely, both en- membrane were observed in the cell body of zymes utilize a glutamic acid as a modification TTLL1 and TTLL9 double knockout strains. A GFP site, and in both cases the target residue is present fusion of Ttll1p localized to basal bodies while within the CTT of tubulin. Subsequent studies Ttll9p was found in both basal bodies and cilia. showed that both E-ligase and G-ligase indeed are Both of these enzymes primarily modify D-tubulin members of the TTLL superfamily. TTLL1, and (Wloga et al., 2008). Thus, glutamylation on D- several other members of the TTLL superfamily tubulin, while clearly not essential, could be im- including TTLL4, TTLL6 and TTLL9 are catalytic portant in regulation of the fidelity of basal body subunits with tubulin E-ligase activity (Ikegami et maturation. A mutation in PGs1, a protein associ- al., 2006; Janke et al., 2005; van Dijk et al., 2007; ated with TTLL1 E-ligase in the mouse causes Wloga et al., 2008). Gene knockouts or knock- severe defects in assembly of sperm axonemes downs of mRNA encoding specific TTLL E- (Campbell et al., 2002). In Tetrahymena, knock- ligases or associated proteins greatly decrease the outs of genes encoding of multiple paralogs of levels of tubulin glutamylation on subsets of TTLL6 type enzyme led to shortening of cilia and microtubules in diverse models: mouse, zebrafish loss of ciliary motility (S. Suryavanshi and JG, and Tetrahymena (Ikegami et al., 2007; Janke et unpublished data). Among these enzymes, al., 2005; Pathak et al., 2007; Wloga et al., 2008). Ttlll6Ap has been well characterized biochemi- Furthermore, in one case, of the murine TTLL7 cally and found to have an exclusive side chain enzyme (that belongs to the TTLL6 clade) it was elongase activity that is largely restricted to E- possible to reconstitute the in vitro tubulin gluta- tubulin (Janke et al., 2005; van Dijk et al., 2007). mylation reaction using a recombinant E-ligase GFP-Ttll6Ap localizes mainly to cilia (Janke et al., (Ikegami et al., 2006; Mukai et al., 2009). 2005). Thus, selective localization of side chain (Ikegami et al., 2006; Janke et al., 2005; Pathak et elongases could be a major mechanism that is re- al., 2007. The clades of E-ligases are fairly well sponsible for differential side chain length distri- conserved across eukaryotes, indicating that sub- bution among distinct types of microtubules. A classes of E-ligases exist that differ in some con- morpholino knockdown of TTLL6 homolog in 12 原生動物学雑誌 第43巻 第1号 2010年 zebrafish leads to shortening of olfactory cilia acts as an elongase for tubulin, could also have (Pathak et al., 2007). Thus, tubulin glutamylation some initiation activity. In addition to a mild re- has emerged as a major factor that contributes to duction in the length of cilia, TTLL3 Tetrahymena the assembly and function of cilia and basal bod- knockout cells elevated levels of acetyl-K40 on D- ies. tubulin of axonemes (Wloga et al., 2009). It thus Recently, enzymes that catalyze glycylation, G- appears that TTLL3-mediated tubulin glycylation ligases, were identified among the previously un- makes axonemal microtubules more dynamic, studied members of the TTLL protein superfamily. which in turn could be a requirement for elonga- Rogowski and colleagues showed that murine tion of axonemes to a proper length. This result fits TTLL3 and TTLL10 act as G-ligases on tubulin. In well with our observation that katanin, a factor that cultured mammalian cells, the murine TTLL3 initi- promotes microtubule turnover, is required for ates glycine side chains that are subsequently elon- ciliogenesis (Sharma et al., 2007). While the im- gated by TTLL10 (Rogowski et al., 2009). Our pact of TTLL3 on the axoneme length was mild in laboratory has identified a TTLL3 type protein of Tetrahymena, this enzyme appears to be important Tetrahymena, Ttll3Ap, as a tubulin G-ligase with for axoneme assembly in multicellular organisms. strong initiating activity (Wloga et al., 2009). The In zebrafish, a morpholino knockdown of TTLL3 fruit fly, Drosophila melanogaster is an interesting led to dramatic shortening of multiple classes of case, because this species lacks an obvious cilia (Wloga et al., 2009) and RNAi-mediated TTLL10 elongase homolog and TTLL3 type pro- knockdown of TTLL3 in Drosophila inhibited teins of Drosophila have both chain initiation and assembly of sperm axonemes (Rogowski et al., elongation G-ligase activity on tubulin (Rogowski 2009). et al., 2009). In Tetrahymena and Drosophila, Another clue to the function of tubulin glycyla- TTLL3 enzymes are required for tubulin glycyla- tion is that Tetrahymena strains lacking TTLL3 tion in vivo (Rogowski et al., 2009; Wloga et al., genes have greatly increased levels of tubulin glu- 2009). tamylation on axonemal and cortical microtubules Tetrahymena has 6 genes encoding TTLL3 type (Wloga et al., 2009). Additional experiments have enzymes. When mildly overexpressed as GFP fu- confirmed that the two polymodifications nega- sions, some TTLL3 paralogs colocalize with cilia, tively regulate each other. Thus, overexpression of some with basal bodies or remain in the cell body E-ligases increased the levels of tubulin glutamyla- (Wloga et al., 2009). Surprisingly, deletion of all tion (as expected) but decreased the levels of tubu- six TTLL3 paralogs resulted in viable cells that are lin glycylation on the same microtubules (Wloga et motile and have normal morphology. However al., 2009). In a reverse experiment, overexpressing cilia in cells lacking TTLL3 activity are on aver- a TTLL3 G-ligase increased the levels of tubulin age 15% shorter as compared to wildtype cells. glycylation and decreased the levels of tubulin The TTLL3 Tetrahymena knockout cells have glutamylation (Wloga et al., 2009). It thus appears residual tubulin glycylation estimated to be be- that the two modifications compete with each other tween 1-8% of wildtype (Wloga et al., 2009). in vivo. The competition could be a result of utili- Since all TTLL3 G-ligases were eliminated in zation of the same Es in the CTT domain, or steric these strains, another tubulin G-ligase with an ini- inhibition of adjacent polymodification sites. tiating activity must exist. This enzyme could be- While tubulin glutamylation is highly conserved long to less conserved classes of TTLLs whose and present in all eukaryotes that also have cilia members are present in Tetrahymena (TTLL14, and centrioles, a few lineages (e.g. nematodes and TTLL15 and TTLL16) or TTLL10 enzyme that trypanosomes) lack tubulin glycylation. It is possi- Jpn. J. Protozool. Vol. 43, No. 1. (2010) 13 ble therefore that the main role of tubulin glycyla- oviduct cells. Biol.Cell. 71:227-245. tion is to negatively regulate tubulin glutamylation Arce, C.A., J.A. Rodriguez, H.S. Barra, and R. and that organisms that lack tubulin glycylation Caputo. 1975. Incorporation of L-tyrosine, L- could have evolved additional mechanisms for phenylalanine and L-3,4-dihydroxyphenylala- negative regulation of tubulin glutamylation. nine as single units into rat brain tubulin. Eur J Biochem. 59:145-9. Argarana, C.E., C.A. Arce, H.S. Barra, and R. SUMMARY Caputto. 1977. In vivo incorporation of [14C] tyrosine into the C-terminal position of the Recent studies brought major advances in eluci- alpha subunit of tubulin. Arch Biochem Bio- dation of the mechanisms that diversify micro- phys. 180:264-8. tubules at the subcellular level. Tubulin polymodi- Argarana, C.E., H.S. Barra, and R. Caputto. 1978. fications have emerged as a major determinant of Release of [14C]-tyrosine from tubulinyl-[14C] assembly and functions of specific microtubules, -tyrosine by brain extract. Separation of a and in particular, those forming cilia and basal carboxypeptidase from tubulin tyrosine ligase. bodies. Studies in ciliates have contributed to iden- Mol. Cell Biochem. 19:17-22. tification of enzymes that polymodify tubulin and Aury, J.M., O. Jaillon, L. Duret, B. Noel, C. Jubin, uncovered complex regulatory interactions among B.M. Porcel, B. Segurens, V. Daubin, V. diverse tubulin modifications. Anthouard, N. Aiach, O. Arnaiz, A. Billaut, J. Beisson, I. Blanc, K. 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