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Functional segregation and emerging role of cilia-related cytosolic carboxypeptidases (CCPs) Mónica Rodríguez de la Vega Otazo,*,† Julia Lorenzo,*,† Olivia Tort,*,† Francesc X. Avilés,*,†,1 and José M. Bautista‡,§,1 *Institute of Biotechnology and Biomedicine and †Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona, Barcelona, Spain; and ‡Department of Biochemistry and Molecular Biology IV, Facultad de Veterinaria, and §Instituto de Investigación Hospital 12 de Octubre, Universidad Complutense de Madrid, Madrid, Spain Recent experimental data indicating axonal regeneration, axogenesis, dendritogenesis, and ciliary axoneme assembly and wellness have linked the role of cytosolic metallocarboxypeptidase 1 (CCP1/ AGTPBP1/Nna1) to the microtubule network. In addition, 5 of the 6 mammalian ccp genes have been shown to participate in post-translational modifications of tubulin, which occur in the microtubules of neurons, mitotic spindles, cilia, and basal bodies. Here, we compile evidence for the idea that the occurrence of CCPs strongly correlates with the presence of cilia, suggesting that CCP functions might be primarily related to cilia and basal bodies (CBBs). In agreement with this hypothesis, CCPs were localized in centrioles, basal bodies, and mitotic spindles in HeLa cells by confocal microscopy. By reconstructing the evolutionary history of CCPs, we show their presence in the last eukaryotic common ancestor and relate each group of CCP orthologs to specific roles in CBBs. The clues presented in this study suggest that during the evolution of eukaryotes, mechanisms mediated by CCPs through tubulin post-translational modifications controlling assembly, trafficking, and signaling in the microtubules, were transferred from cilia to cell and axon microtubules.—Rodríguez de la Vega Otazo, M., Lorenzo, J., Tort, O., Avilés, F. X., Bautista, J. M. Functional segregation and emerging role of cilia-related cytosolic carboxypeptidases (CCPs). FASEB J. 27, 424 – 431 (2013). www.fasebj.org ABSTRACT

Key Words: basal bodies 䡠 microtubules 䡠 tubulin modifications 䡠 polyglutamylation 䡠 detyrosination

Abbreviations: 3D, 3-dimensional; AGBL, ATP/GTP binding protein-like; AGTPBP1, ATP/GTP binding protein 1; CBB, cilia and basal body; CCP, cytosolic metallocarboxypeptidase; DAPI, 4=,6-diamidino-2-phenylindole; IFT, intraflagellar transport; LECA, last eukaryotic common ancestor; Nna1, nervous system nuclear protein induced by axotomy 1; pcd, Purkinje cell degeneration; PTM, post-translational modification; SSA, sensory, structural, and assembly; tM, Manders’ coefficient with threshold; TTL, tubulin tyrosine ligase; TTLL, tubulin tyrosine ligase-like; tubCP, tubulin carboxypeptidase 424

Post-translational modifications (PTMs) affecting tubulin occur in the microtubules of neurons, mitotic spindles, and cilia and basal bodies (CBBs). PTMs play an important role in the dynamics and organization of microtubules and thus have a direct effect on cell functioning and cilia wellness, which are critical in human health and disease (1–3). Cytosolic metallocarboxypeptidases (CCPs), a recently proposed novel subfamily of peptidases (4, 5), are known to cooperate in the PTM events that take place at the C-terminal tail of tubulin (detyrosination, generation of ⌬2-tubulin, and polyglutamylation) together with tubulin tyrosine ligase (TTL) and tubulin tyrosine ligase-like (TTLL) enzymes (6 – 8). In particular, mammalian CCPs 1, 4, 5, and 6 play a role in the polyglutamylation of tubulin (6, 8); CCPs 1, 4, and 6 contribute to the generation of ⌬2-tubulin (6), and CCP2 has been reported to act in the detyrosination of ␣-tubulin (7). Although some advances have been made in the field of CCP specificities, the biological processes in which these enzymes act remain largely unknown, with the exception of the role played by CCP1 [ATP/GTP binding protein 1 (AGTPBP1), nervous system nuclear protein induced by axotomy 1 (Nna1)]. Over the past 10 yr, experimental evidence has emerged that links the functions of CCP1 with the microtubule network. In early work, it was determined that mouse ccp1 is up-regulated during axon reinervation and is overexpressed during axogenesis (9). Subsequent to this, ccp1 was shown to be responsible for the Purkinje cell degeneration (pcd) phenotype in mice (10). These mice also exhibit photoreceptor degeneration, defective spermatogenesis (11), and deficient assembly of axonemal and periaxonemal structures 1 Correspondence: J.M.B., Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain. E-mail: jmbau@ vet.ucm.es; F.X.A, Institute of Biotechnology and Biomedicine, Universitat Autonoma de Barcelona, Bellaterra (Barcelona), Spain. E-mail: [email protected] doi: 10.1096/fj.12-209080 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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within the sperm flagellum (12). In C. elegans, loss of CCP1 function causes progressive deterioration of the ciliary axoneme (13). Moreover, RNAi-mediated knockdown of CCP1 in cultured human cells induces the reduction of ciliary length (14). Thus, these prior reports appear to link the functions of CCP1 orthologs not only to axon microtubules but also to ciliary structures. Much more meager is the information available on the other CCPs (i.e., CCP2 to CCP6 in mammals), which are all cytosolic and/or nuclear in eukaryotes. In previous work, we ascribed these CCPs to a new subfamily (5) that seems to share localizations and roles (3– 8). Cilia are not only restricted to flagellated sperm or ciliated receptors. Indeed, primary cilia are key organelles in numerous physiological and developmental processes. Defects in ciliary function lead to a wide range of syndromes, called ciliopathies (2). Accordingly, interest mounts in gaining insight into the structural and dynamic components of cilia to clarify their dysfunctions. Given that pcd mice show clear ciliopathy signs and that CCP1 is known to affect ciliogenesis and cilia well-being, we hypothesized that a primary function of CCPs could be related to cilia and basal bodies. Then, throughout evolutionary history, the mechanisms of signaling to the microtubule network through post-translational modifications of tubulin would be eventually transferred from cilia to mitotic spindles and to axon microtubules. To strengthen our hypothesis by providing clues on the bigger picture, we searched for CCP genes in genome databases of representative ciliated and nonciliated organisms. It was found that CCPs occur only in organisms that have cilia, including the last eukaryotic common ancestor (LECA). With this new perspective, we established ortholog/paralog relationships for CCP genes and, based on the available information regarding the microtubule biology of organisms with CCPs, we propose new functions associated with microtubular and cilia-related structures for each CCP cluster. Finally, by confocal microscopy, we localize CCP members in cilia-related structures and in the mitotic spindle of dividing cells. MATERIALS AND METHODS Dataset definition Database searches for CCP genes were conducted as described previously (5). Sequences from 31 ciliated and 16 nonciliated eukaryotic organisms spanning 6 major clades and with a rich diversity of microtubule biology were selected as described elsewhere (15, 16). A normalized Hamming distance, defined as 100 ⫻ [number of species showing correlation/total number of species] (15) was used to calculate correlation between the presence of CCPs and CBBs. Phylogenetic analyses A total of 147 CCP sequences (Supplemental Table S3) were aligned using T-Coffee (17). The tridimensional structure of CILIA-RELATED CYTOSOLIC CARBOXYPEPTIDASES

the CCP recently released (18) led us to define the conserved core of CCPs and consequently to refine the sequencestructural alignment of CCPs used in the phylogenetic analyses. The alignment was manually corrected, taking into account the structural alignment information described previously (5). The definitive alignment length was 141 sites. A set of sequences, for which phylogenetic inferences were stable regardless of the method used, was first established. The remaining sequences were introduced one by one, and their position on the tree was recorded. Trichomonas vaginalis sequences XP_001580022.1 and XP_001581178.1 (M14D3) were excluded from phylogenetic analysis due to long-branch attraction. Phylogenetic reconstructions were obtained using Molecular Evolutionary Genetics Analysis 4 (MEGA4; ref. 19). Branch lengths were estimated by the neighbor-joining (20) and minimum evolution (21) methods under the Poisson correction model (22). Bootstrapping (23) and the interior branch test (1000 replicates; ref. 24) were conducted for each tree topology obtained. Trees were plotted using the MEGA4 Tree Explorer. Cell culture, immunofluorescence, and microscopy HeLa or NIH-3T3 cells were plated on glass coverslips treated with 0.01% sterile polylysine. Sixty percent confluent cultured cells were washed with PBS, fixed in cold methanol for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked for 1 h with 5% BSA in PBS containing 0.05% Tween. Cells were stained with primary antibodies overnight in the cold. The primary antibodies used for immunofluorescence were as follows: mouse anti-␣-tubulin clone DM1A (Sigma, St. Louis, MO, USA), mouse antigolgina GM130 (BD Bioscience, San Jose, CA, USA), mouse anti-␥-tubulin clone Tu-30 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-monoglutamylated-tubulin clone GT335 (Enzo, Farmingdale, NY, USA), rabbit anti-AGTPBP1/ CCP1 (ProteinTech Group, Chicago, IL, USA), rabbit antiATP/GTP binding protein-like 2 (AGBL2)/CCP2 (Sigma), rabbit anti-AGBL4/CCP6 (Abcam, Cambridge, MA, USA), and rabbit anti-AGBL5/CCP5 (Abcam). Cells were then washed and incubated with secondary antibodies for 1 h using AlexaFluor488-conjugated anti-mouse and AlexaFluor555- or 647-conjugated anti-rabbit (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4=,6-diamidino-2-phenylindole (DAPI; Sigma). Coverslips were mounted with FluoPrep antifade reagent. Images were acquired at room temperature on a Leica TCS SP5 confocal microscope using an ⫻63 (1.4) oil-immersion objective (Leica Microsystems, Wetzlar, Germany). ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; ref. 25) was used for image smoothing using a gaussian filter. Manders’ coefficients (tM1 and tM2), ranging from 0 (total exclusion) to 1 (total colocalization) were calculated using “just another colocalization plugin” in ImageJ with automatic threshold (26). For 3-dimensional (3D) reconstructions by surface rendering, adjusting cutoff threshold values to remove black voxels from rendering, we used Imaris 7.2.3 (Bitplane, Zurich, Switzerland).

RESULTS Evidence for the presence of CCPs in LECA After an extensive search for CCP genes in databases, we found that prokaryotic CCPs are restricted to a reduced number of bacteria (Fig. 1 and Supplemental Table S1) while eukaryotic CCPs are widely distributed in 425

Figure 1. Taxonomic distribution of CCPs. Homologous CCP genes were identified in bacteria and in the major groups of eukaryotes by genome scanning suggesting their presence in the LECA. Eukaryotic CCPs are widely distributed in holozoans and protists and in the basal lineages of plants and fungi. Background image from the Tree of Life Web Project © 2007, licensed under Creative Commons Version 3.0, following their established rules.

holozoans (animals, placozoans, and choanoflagellates) and protists, although only found in the basal lineages of plants (Chlorophyta: Chlamydomonas reinhardtii and Volvox carteri) and fungi (Chytridiomycota: Batrachochytrium dendrobatidis; Fig. 1 and Supplemental Table S2). Thus, the presence of CCPs in the major groups of eukaryotes suggests their occurrence in the LECA. The most widely accepted endosymbiotic models explaining the origin of eukaryotic cells postulate the fusion or association of distinct bacterial and archaeal partners. Such postulates consider two plausible models: the coorigin of mitochondrion and nucleus, and the “nucleus first–mitochondria later” hypothesis (27). In the above-mentioned search, it was also found that CCP genes occur in the amitochondrial protists Giardia lamblia and Trichomonas vaginalis, and their presence in flagellated green algae are exceptions among the large number of chloroplast-bearing organisms (Figs. 1 and 2 and Supplemental Table S2). Thus, we were unable to correlate the presence of CCP genes with the presence of mitochondria or plastids, suggesting that eukaryotic CCP genes were acquired before the endosymbiotic events that gave rise to mitochondria and chloroplasts in eukaryotic cells. This is more in line with the nucleus first–mitochondria later model. According to this model, the ancestral eukaryotic cell arose through the 426

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fusion and integration of a bacterium and an archae prior to the acquisition of mitochondria or plastids (27). Extensive screening of prokaryote databases revealed CCP sequences in Bacteria but not in Archae (Fig. 1), suggesting that eukaryotic CCPs were acquired through the bacterial partner in the first fusion. Notably, the fused bacterium has been hypothesized to be either a spirochaete (28) or a gram-negative bacterium related to proteobacteria (29, 30). We found that bacterial CCPs are mainly distributed in proteobacteria but absent in spirochaetes (Fig. 1 and Supplemental Table S1), supporting the hypothesis that the bacterial partner of the first fusion was a proteobacterium. CCPs are distributed among ciliated organisms CBBs are conserved in most eukaryotic groups, suggesting that they were present in the LECA (31) but subsequently lost in fungi and higher plants (15). We observed that the taxonomic distribution of eukaryotic CCP genes (holozoans, protists, and basal lineages of fungi and plants; Fig. 1) is similar to that of CBBs. Consequently, we explored whether the presence of CCPs correlates with the occurrence of these structures (Fig. 2). With the use of the normalized Hamming distance method, ⬎95% correlation was detected be-

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deficient in genes encoding intraflagellar transport proteins (34). However, the ciliated diatom Thalassiosira pseudonana (which has a single CCP gene ortholog to that of P. tricornutum) builds motile flagellated gametes within the cytoplasm environment, suggesting a high versatility of diatom CCP function. Having established that the distribution of CCPs positively correlates with the presence of cilia and basal bodies, we propose that CCPs belong to a set of ancestral proteins with a role in building up these structures and their associated cell functions. Since prokaryotic flagella do not share structures with eukaryotic cilia (in terms of basal bodies and participating structural proteins: e.g., flagellin vs. tubulin), we excluded bacterial CCP proteins from our analysis. Segregation of CCP functions into paralog genes

Figure 2. Occurrence of CCP genes in representative ciliated and nonciliated organisms among the main eukaryotic groups: Plantae (plants), Protista (Excavata and Chromalveolata), Holozoa, Fungi, and Amoebozoa (Am.). Nonciliated organisms are highlighted in gray. Figure shows a drawing of each organism, the number of CCP paralogs, and the number of orthologs belonging to the M14D group defined by the phylogenetic analysis shown in Fig. 3.

tween the presence of CCPs and CBBs spanning the evolution of eukaryotes. All the ciliated organisms analyzed had CCP genes, with the exception of the lower plant Physcomitrella patens (Fig. 2). Although this plant produces motile flagellate zoospores, it lacks sets of centriolar-related genes, which in mammals are associated with ciliopathies such as Bardet-Bield syndrome and Meckel-Gruber syndrome (16). Moreover, no CCP genes were identified in nonciliated organisms, with the exception of the diatom Phaeodactylum tricornutum (Fig. 2). Diatoms have no centrioles (32) or corresponding structural proteins (16, 33) and are CILIA-RELATED CYTOSOLIC CARBOXYPEPTIDASES

In an attempt to segregate the different CCP functions into their homologue genes, we first defined CCP orthologs by phylogenetic analysis of their peptidase domain (Fig. 3 and Supplemental Table S3). Next, we mapped the potential functions of CCP orthologs according to their occurrence either in organisms containing only primary cilia (C. elegans) or only motile flagella (e.g., B. dendrobatidis) to correspondingly assign them a sensory, structural, and assembly (SSA) or motile function. We also considered the available information on the biology and substrate specificity of CCPs. Finally, we examined the intracellular localization of CCPs in HeLa and NIH-3T3 cells to further support our functional assignments, focusing on representatives of the main clades indicated by phylogenetics (Fig. 4). Since C. elegans lacks motile cilia and CCP5 genes (Fig. 3, M14D2/CCP5), we inferred that CCP5 orthologs have motile functions. Mouse CCP5 has been reported to preferentially cleave branching glutamate from post-translationally modified tubulin (6). Moreover, inhibition of tubulin polyglutamylation leads to increased cilia speed (35). Thus, controlling branching points in axoneme microtubules via CCP5 deglutamylase activity may directly regulate tubulin polyglutamylation and, consequently, cilia motility. We characterized the intracellular localization of CCP5 in HeLa cells and found that human CCP5 changes during the cell cycle (Fig. 4A–C). In nondividing cells, CCP5 colocalized with the nuclei marker DAPI but not with ␣-tubulin (Fig. 4A). In contrast, the CCP5 signal was concentrated at the mitotic spindle during the different steps of cell division (Fig. 4B). In metaphase, 63% of the CCP5 signal overlapped the ␣-tubulin signal (tM1: 0.632). The fraction of ␣-tubulin overlapping the CCP5 signal was 0.908 (tM2), indicating that almost all ␣-tubulin colocalized with CCP5 in metaphase. As the cell cycle progressed, tubulin and CCP5 colocalization diminished (tM2: 0.784 in anaphase and 0.308 in telophase). In addition, CCP5 was observed to colocalize with midbody microtubules in the intercellular bridges formed during cytokinesis (Fig. 4C). It has been reported that mitotic spindle microtubules are glutamy427

Figure 3. Evolutionary relationships among cytosolic carboxypeptidases. Evolutionary history of CCPs was phylogenetically inferred. Tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Different groups of orthologs are highlighted in different colors. Prokaryote sequences cluster in M14D1a/b and eukaryote sequences in M14D1b, D2, D3, and D4. Numbers at nodes are frequency values obtained in the interior branch test (1000 replicates) using the neighbor-joining method under the Poisson correction model.

lated. In HeLa cells, they are mainly monoglutamylated at the ␤-tubulin subunit (36). As CCP5 preferentially hydrolyzes branching glutamates, this enzyme may regulate tubulin glutamylation at the level of the mitotic spindle, playing a role in cell division. Further experimental evidence is needed to confirm this hypothesis. In effect, clues on its functional role may be provided by characterizing substrate specificity using purified enzymes. The proteins found to cluster with C. elegans CCP6 (Fig. 3, M14D2/CCP6) would be expected to show SSA but not motile function. Human CCP6 generates ⌬2tubulin and shows deglutamylase activity in long polyglutamylated chains (6). Accordingly, CCP6 orthologs might act at the CBB level to generate ⌬2-tubulin or control the length of polyglutamylated chains in microtubules forming cilia axoneme or basal body triplets. In support of our hypothesis that CCP6 shares SSA functions, the protein has been recently localized in ciliated sensory neurons in C. elegans, and its overexpression reduces the monoglutamylation signal in sensory cilia microtubules (8). Further, we detected the presence of 428

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CCP6 in Naegleria gruberi (Fig. 2 and Supplemental Table S3), which changes from an amoeba into a flagellate by de novo assembly of an entire cytoplasmic microtubule cytoskeleton, including canonical basal bodies and flagella (37). Since CCPs were absent in Amoebozoa (Figs. 1 and 2), the presence of CCP6 in N. gruberi is linked to its flagellated state. N. gruberi has motile flagella but lacks primary cilia. Consequently, CCP6 orthologs may play a role in the de novo assembly of CBB and have an SSA function not restricted to primary cilia. The distribution of CCP6 in HeLa and NIH-3T3 cells was also determined (Fig. 4D–F). CCP6 was found in the Golgi apparatus (Fig. 4D) and centrioles (Fig. 4E) of interphase cells. In mitotic cells, the CCP6 signal was stronger in centrioles and colocalized with ␥-tubulin (Fig. 4E). The localization of CCP6 in the Golgi complex is not necessarily a conflicting observation, since this organelle and the cilia are closely related structurally and functionally (38). In addition, several intraflagellar transport (IFT) proteins are localized in the Golgi apparatus, such as the motor proteins kinesin 2 (39) and dynein 2 (40), as well as

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Figure 4. Intracellular localization of CCPs. This was done by confocal microscopy and staining with specific antibodies. A, B) CCP5 (red) was localized in the nuclei (blue) of nondividing HeLa cells (A) and colocalized with ␣-tubulin (green) in the mitotic spindle during cell division (B). C) Colocalization of CCP5 and ␣-tubulin remains in intercellular bridges. A colocalization overlay (white points) is shown. D, E) CCP6 (red) colocalized with Golgina GM130 (green) in the Golgi complex (D), and with ␥-tubulin (green) in the centrioles at interphase and dividing HeLa cells (E). F) CCP6 colocalized with glutamylated tubulin (green) in basal bodies of ciliated NiH-3T3 cells. G) CCP1 (red) localized in both the cytoplasm and nuclei (blue) of interphase and dividing HeLa cells. H, I) CCP2 (red) colocalized with ␥-tubulin (green) in the centrioles of HeLa cells (H) and with glutamylated tubulin (green) in the basal bodies of ciliated HeLa cells (I). The 3D reconstructions shown in panels D, F and I were prepared using Imaris software. Scale bars ⫽ 20 ␮m (A); 5 ␮m (B–E, G, H); 1 ␮m (F, I).

IFT20, which is highly dynamic and moves from the Golgi system to the cilium (41). Moreover, we detected CCP6 in the basal bodies of ciliated NIH-3T3 cells (Fig. 4F). Thus, the cellular localization of CCP6 is consistent with the SSA function that we predict for CCP6 orthologs. Notwithstanding, the specific mechanisms underlying CCP6 functioning within these organelles and crosstalk with other associated proteins needs to be examined by specific cell biology approaches. The C. elegans protein O76373 clustered with M14D3 genes, suggesting an SSA function for this set of orthologs. Human CCP1 and CCP4 paralogs were also found to cluster in the M14D3 clade (Fig. 3). CCP1 function has been linked to ciliogenesis and the well being of primary cilia through its actions as a positive modulator of ciliogenesis in retinal pigment epithelium cells (14). Our confocal microscopy analysis of CCP1 in HeLa cells confirms its previously reported localization in the nucleus and cytoplasm, showing a granular distribution in interphase and dividing cells (Fig. 4G). The nuclear fraction of CCP1 could be involved in DNA repair processes, as the pcd mutation induces DNA damage/repair foci in degenerating mitral cells (42). CILIA-RELATED CYTOSOLIC CARBOXYPEPTIDASES

At the cytoplasm level, the loss of CCP1 function in pcd mice leads to the partial replacement of rough endoplasmic reticulum cisternae by a larger amount of free ribosomes (42). The lack of CCP1 either in the mouse or C. elegans causes the degeneration of photoreceptors (11) or progressive deterioration of ciliary axonemes (13), respectively. However, CCP1 is also involved in motile cilia functions since the pcd mouse (lacking CCP1 activity) displays defective spermatogenesis (11), failing to assemble axoneme and periaxoneme structures (10, 12). Mouse CCP1 participates in axogenesis and axonal regeneration (9) and acts by shortening polyglutamate side chains in both alpha and beta tubulin and generating ⌬2-tubulin in brain microtubules (6, 43, 44). Moreover, CP1 can generate an additional form of ␣-tubulin lacking 2 C-terminal Glu residues (⌬3-tubulin; ref. 43). Thus, the generation of ⌬2/⌬3-tubulin and the hydrolysis of polyglutamylated chains, i.e., the primary enzyme functions of CCP1 (43), seem to be essential in nerve reinervation and the correct assembly of axoneme microtubules. In addition, the only CCP gene identified in B. dendrobatidis was also noted to cluster 429

within the M14D3 clade. This organism lacks primary cilia and produces motile uniflagellate zoospores (45). Accordingly, the functions of M14D3 genes could be associated with the assembly and elongation of the microtubule network, which takes place at the CBB level in the building of primary or motile ciliary axonemes and at the axonal level during reinervation and axogenesis. M14D4 peptidases (clustering with human CCP2 and CCP3 paralogs) are absent in C. elegans (Fig. 3, M14D4) and as such, their main functions may be associated with motile cilia. Human CCP2 has been reported to act as tubulin carboxypeptidase (tubCP; ref. 7) In agreement, C. elegans lacks the TTL gene (46), which acts in the detyrosination/retyrosination cycle of tubulin together with tubCP (47). However, the Drosophila melanogaster genome encodes an M14D4 gene (Fig. 3) but lacks the TTL gene (46), which seems incongruent with M14D4-tubCP activity. Two TTLL genes acting in tubulin PTMs as polyglycylases have been described in D. melanogaster (48) and are absent in C. elegans (46, 48). Notably, the enzymes acting as deglycylases in holozoans remain unknown, but recently two enzymes belonging to the M20 family of peptidases, which display deglycylase activity on the multifunctional proteins 14-3-3 in Giardia duodenalis, have been identified (49). Interestingly, our recently resolved structure of the Pseudomona aeruginosa CCP (18) is related to M20 peptidases. Finally, human CCP2 found in the basal bodies of ciliated HeLa cells colocalized with ␥-tubulin across all stages of the cell cycle (Fig. 4H, I). This localization of CCP2 in centrioles and basal bodies of ciliated and nonciliated HeLa cells supports their role in CBB functioning. Further research will be required, however, to clearly confirm and clarify such assignments and functions proposed here at the hypothesis level. The above phylogenetic mapping of CCP functions clearly demonstrates strong correlation between the evolution of basal bodies and cilia and the presence of different CCP enzymes, underlying the prominent role of tubulin modifications in basal bodies, cilia, and flagella. Nevertheless, at present there is no further experimental evidence associating the specific catalysis of single CCP enzymes and their phylogenetic distribution. The phylogenetic clustering of other enzymes involved in tubulin post-translational modifications has not always been sufficient to predict their enzyme specificities. For example, mouse TTLL6 polyglutamylase specific to ␣-tubulin has a close homologue in Tetrahymena (TTLL6A) that is highly specific to ␤-tubulin (50). Thus, tubulin-modifying enzymes might have undergone the relatively rapid adaptation of their specificities in different species reflecting the evolutionary flexibility of their functions. CONCLUSIONS

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The authors thank Dr. Luis Serrano (Centre de Regulació Genòmica, Barcelona, Spain), Jesús Avila (Centro de Biología Molecular–Consejo Superior de Investigaciones Cientificas, Madrid, Spain), and Oscar Yanes (Universitat Rovira i Virgili, Tarragona, Spain) for discussions and reading of the manuscript; M. Roldán and N. Barba for technical assistance with confocal microscopy imaging and analyses; and F. Cortés for technical support in cell culturing. This work was funded by grants from the Spanish Ministry of Science and Innovation (BIO2010-22321 and BIO2010-17039), by the Network of Excellence of the Generalitat de Catalunya (Xarxa de Referència en Biotecnologia; Spain), by the Iberoamerican Ciencia y Tecnología Para el Desarrollo (CYTED) network (210RT0398), and by the Program of Consolidated Research Teams of the Universidad Complutense de Madrid (J.M.B., Research Team 920267; Comunidad de Madrid, Spain).

REFERENCES 1. 2. 3. 4. 5.

6.

7.

8.

9.

This functional segregation survey of CCPs across eukaryotic evolution highlights their ancient tasks related 430

to CBBs. Thus, early in evolution, associated CCP trafficking and signaling mechanisms were transferred from CBBs to both cell microtubules, to acquire a new role in the cell cycle, and to axon microtubules, for the genesis and regeneration of neurons. The evolutionary history of CCPs opens a wide spectrum of possibilities for future studies designed to unravel protein-substrate specificity and homologous gene recruitment for functional diversification in many different organisms. Both these factors play critical roles in human health and disease. Eventually, our hypothesized functional segregation of CCPs should be amenable to empirical analysis in model cells across different ciliogenesis stages following experimental knockdown or overexpression of CCP orthologs.

Wloga, D., and Gaertig, J. (2010) Post-translational modifications of microtubules. J. Cell Sci. 123, 3447–3455 Ishikawa, H., and Marshall, W. F. (2011) Ciliogenesis: building the cell’s antenna. Nat. Rev. Mol. Cell Biol. 12, 222–234 Janke, C., and Bulinski, J. C. (2011) Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat. Rev. Mol. Cell Biol. 12, 773–786 Kalinina, E., Biswas, R., Berezniuk, I., Hermoso, A., Aviles, F. X., and Fricker, L. D. (2007) A novel subfamily of mouse cytosolic carboxypeptidases. FASEB J. 21, 836 –850 Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., and Aviles, F. X. (2007) Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily. FASEB J. 21, 851–865 Rogowski, K., van Dijk, J., Magiera, M. M., Bosc, C., Deloulme, J. C., Bosson, A., Peris, L., Gold, N. D., Lacroix, B., Grau, M. B., Bec, N., Larroque, C., Desagher, S., Holzer, M., Andrieux, A., Moutin, M. J., and Janke, C. (2010) A family of proteindeglutamylating enzymes associated with neurodegeneration. Cell. 143, 564 –578 Sahab, Z. J., Hall, M. D., Me Sung, Y., Dakshanamurthy, S., Ji, Y., Kumar, D., and Byers, S. W. (2010) Tumor suppressor RARRES1 interacts with cytoplasmic carboxypeptidase AGBL2 to regulate the alpha-tubulin tyrosination cycle. Cancer Res. 71, 1219 –1228 Kimura, Y., Kurabe, N., Ikegami, K., Tsutsumi, K., Konishi, Y., Kaplan, O. I., Kunitomo, H., Iino, Y., Blacque, O. E., and Setou, M. (2010) Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs). J. Biol. Chem. 285, 22936 –22941 Harris, A., Morgan, J. I., Pecot, M., Soumare, A., Osborne, A., and Soares, H. D. (2000) Regenerating motor neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases. Mol. Cell. Neurosci. 16, 578 –596

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10.

11. 12. 13.

14.

15.

16. 17.

18.

19. 20. 21.

22. 23. 24. 25. 26. 27 28. 29. 30. 31. 32. 33. 34.

Fernandez-Gonzalez, A., La Spada, A. R., Treadaway, J., Higdon, J. C., Harris, B. S., Sidman, R. L., Morgan, J. I., and Zuo, J. (2002) Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1. Science 295, 1904 –1906 Mullen, R. J., Eicher, E. M., and Sidman, R. L. (1976) Purkinje cell degeneration, a new neurological mutation in the mouse. Proc. Natl. Acad. Sci. U. S. A. 73, 208 –212 Escalier, D. (2006) Knockout mouse models of sperm flagellum anomalies. Hum. Reprod. Update. 12, 449 –461 O’Hagan, R., Piasecki, B. P., Silva, M., Phirke, P., Nguyen, K. C., Hall, D. H., Swoboda, P., and Barr, M. M. (2011) The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans. Curr. Biol. 21, 1685–1694 Kim, J., Lee, J. E., Heynen-Genel, S., Suyama, E., Ono, K., Lee, K., Ideker, T., Aza-Blanc, P., and Gleeson, J. G. (2010) Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464, 1048 –1051 Carvalho-Santos, Z., Machado, P., Branco, P., Tavares-Cadete, F., Rodrigues-Martins, A., Pereira-Leal, J. B., and BettencourtDias, M. (2010) Stepwise evolution of the centriole-assembly pathway. J. Cell Sci. 123, 1414 –1426 Hodges, M. E., Scheumann, N., Wickstead, B., Langdale, J. A., and Gull, K. (2010) Reconstructing the evolutionary history of the centriole from protein components. J. Cell Sci. 123, 1407–1413 Di Tommaso, P., Moretti, S., Xenarios, I., Orobitg, M., Montanyola, A., Chang, J. M., Taly, J. F., and Notredame, C. (2011) T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 39, W13–17 Otero, A., Rodriguez de la Vega, M., Tanco, S., Lorenzo, J., Aviles, F. X., and Reverter, D. (2012) The novel structure of a cytosolic M14 metallocarboxypeptidase (CCP) from Pseudomonas aeruginosa: a model for mammalian CCPs. FASEB J. 26, 3754 –3764 Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596 –1599 Saitou, N., and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406 –425 Rzhetsky, A., and Nei, M. (1992) Statistical properties of the ordinary least-squares, generalized least-squares, and minimumevolution methods of phylogenetic inference. J. Mol. Evol. 35, 367–375 Zuckerkandl, E., and Pauling, L. (1965) Molecules as documents of evolutionary history. J. Theor. Biol. 8, 357–366 Felsenstein, J. (1985) Confidence-limits on phylogenies - an approach using the bootstrap. Evolution 39, 783–791 Dopazo, J. (1994) Estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach. J. Mol. Evol. 38, 300 –304 Collins, T. J. (2007) ImageJ for microscopy. BioTechniques 43, 25–30 Bolte, S., and Cordelieres, F. P. (2006) A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 Gupta, R. S. (2005) Molecular sequences and the early history of life. In Microbial Phylogeny and Evolution: Concepts and Controversies (Sapp, J. ed) pp. 160 –183, Oxford University Press, New York Dolan, M. F., Melnitsky, H., Margulis, L., and Kolnicki, R. (2002) Motility proteins and the origin of the nucleus. Anat. Rec. 268, 290 –301 Moreira, D., and Lopez-Garcia, P. (1998) Symbiosis between methanogenic archaea and delta-proteobacteria as the origin of eukaryotes: the syntrophic hypothesis. J. Mol. Evol. 47, 517–530 Gupta, R. S., and Golding, G. B. (1996) The origin of the eukaryotic cell. Trends Biochem. Sci. 21, 166 –171 Bornens, M., and Azimzadeh, J. (2007) Origin and evolution of the centrosome. Adv. Exp. Med. Biol. 607, 119 –129 Pickett-Heaps, J. D., and Tippit, D. H. (1978) The diatom spindle in perspective. Cell 14, 455–467 Woodland, H. R., and Fry, A. M. (2008) Pix proteins and the evolution of centrioles. PLoS. One. 3, e3778 Armbrust, E. V., Berges, J. A., Bowler, C., Green, B. R., Martinez, D., Putnam, N. H., Zhou, S., Allen, A. E., Apt, K. E., Bechner, M., Brzezinski, M. A., Chaal, B. K., Chiovitti, A., Davis, A. K., Demarest, M. S., Detter, J. C., Glavina, T., Goodstein, D., Hadi, M. Z., Hellsten, U.,

CILIA-RELATED CYTOSOLIC CARBOXYPEPTIDASES

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47. 48.

49.

50.

Hildebrand, M., Jenkins, B. D., Jurka, J., Kapitonov, V. V., Kroger, N., Lau, W. W., Lane, T. W., Larimer, F. W., Lippmeier, J. C., Lucas, S., Medina, M., Montsant, A., Obornik, M., Parker, M. S., Palenik, B., Pazour, G. J., Richardson, P. M., Rynearson, T. A., Saito, M. A., Schwartz, D. C., Thamatrakoln, K., Valentin, K., Vardi, A., Wilkerson, F. P., and Rokhsar, D. S. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306, 79 –86 Suryavanshi, S., Edde, B., Fox, L. A., Guerrero, S., Hard, R., Hennessey, T., Kabi, A., Malison, D., Pennock, D., Sale, W. S., Wloga, D., and Gaertig, J. (2010) Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity. Curr. Biol. 20, 435–440 Regnard, C., Desbruyeres, E., Denoulet, P., and Edde, B. (1999) Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells. Journal of cell science 112(Pt. 23), 4281–4289 Fritz-Laylin, L. K., Assaf, Z. J., Chen, S., and Cande, W. Z. (2010) Naegleria gruberi de novo basal body assembly occurs via stepwise incorporation of conserved proteins. Eukaryot. Cell 9, 860 –865 Tachi, S., Tachi, C., and Lindner, H. R. (1974) Influence of ovarian hormones on formation of solitary cilia and behavior of the centrioles in uterine epithelial cells of the rat. Biol. Reprod. 10, 391–403 Le Bot, N., Antony, C., White, J., Karsenti, E., and Vernos, I. (1998) Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus. J. Cell Biol. 143, 1559 –1573 Vaisberg, E. A., Grissom, P. M., and McIntosh, J. R. (1996) Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J. Cell Biol. 133, 831–842 Follit, J. A., Tuft, R. A., Fogarty, K. E., and Pazour, G. J. (2006) The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol. Biol. Cell 17, 3781–3792 Valero, J., Berciano, M. T., Weruaga, E., Lafarga, M., and Alonso, J. R. (2006) Pre-neurodegeneration of mitral cells in the pcd mutant mouse is associated with DNA damage, transcriptional repression, and reorganization of nuclear speckles and Cajal bodies. Mol. Cell. Neurosci. 33, 283–295 Berezniuk, I., Vu, H. T., Lyons, P. J., Sironi, J. J., Xiao, H., Burd, B., Setou, M., Angeletti, R. H., Ikegami, K., and Fricker, L. D. (2012) Cytosolic carboxypeptidase 1 is involved in processing of alpha-tubulin and beta-tubulin. J. Biol. Chem. 287, 6503–6517 Wu, H. Y., Wang, T., Li, L., Correia, K., and Morgan, J. I. (2012) A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice. [E-pub ahead of print] FASEB J. doi: 10.1096/fj.12-205047 Berger, L., Hyatt, A. D., Speare, R., and Longcore, J. E. (2005) Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Dis. Aquat. Organ. 68, 51–63 Janke, C., Rogowski, K., Wloga, D., Regnard, C., Kajava, A. V., Strub, J. M., Temurak, N., van Dijk, J., Boucher, D., van Dorsselaer, A., Suryavanshi, S., Gaertig, J., and Edde, B. (2005) Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308, 1758 –1762 Barra, H. S., Arce, C. A., and Argarana, C. E. (1988) Posttranslational tyrosination/detyrosination of tubulin. Mol. Neurobiol. 2, 133–153 Rogowski, K., Juge, F., van Dijk, J., Wloga, D., Strub, J. M., Levilliers, N., Thomas, D., Bre, M. H., Van Dorsselaer, A., Gaertig, J., and Janke, C. (2009) Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell 137, 1076 –1087 Lalle, M., Camerini, S., Cecchetti, S., Fantauzzi, C. B., Crescenzi, M., and Pozio, E. (2011) Giardia duodenalis 14-3-3 protein is polyglycylated by a tubulin tyrosine ligase-like member and deglycylated by two metallocarboxypeptidases. J. Biol. Chem. 286, 4471–4484 Van Dijk, J., Rogowski, K., Miro, J., Lacroix, B., Edde, B., and Janke, C. (2007) A targeted multienzyme mechanism for selective microtubule polyglutamylation. Mol. Cell 26, 437–448 Received for publication April 10, 2012. Accepted for publication October 1, 2012.

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