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Purification of tubulin polyglutamylase from Crithidia fasciculata. (A) Affinity chromatography of the 0.25 M salt wash of cytoskeletons (fraction I) on Sepharose-sebacic acid-ATP. At point (1) the column was washed with 0.25 M KCl; at point (2) the enzyme was eluted with 20 mM ATP, 0.5 M KCl. 1 ml fractions were collected and a 2 µl sample of each fraction was assayed for glutamylation activity (fraction II). (B) Further purification of the enzyme by glycerol-gradient centrifugation. 30 fractions were collected from each tube and 10 µl samples were assayed for glutamylation activity (fraction III). The direction of sedimentation is from left to right. The positions of marker proteins are indicated at the top (1: ribonuclease A, 2 S; 2: ovalbumin, 3.6 S; 3: BSA, 4.2 S; 4: aldolase, 7.2 S). (C) Anion-exchange chromatography of the enzyme (fraction III) on a Mono Q column. 2 µl samples were assayed for glutamylation activity (fraction IV). No enzymatic activity was detected in the combined flow-through fractions. The NaCl gradient from 0-0.9 M is indicated as a dashed line. (D) 10% SDS-PAGE of Crithidia tubulin polyglutamylase. Lane 1, 15 µg of crude enzyme (fraction I), stained with Coomassie Brillant Blue; lane 2: approx. 100 ng of the enzyme after anion-exchange chromatography (fraction IV), silver-stain. Relative molecular masses of standard proteins are indicated.
Source publication
Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzyma...
Contexts in source publication
Context 1
... solubilised enzyme (fraction I) was subjected to ATP- affinity chromatography (Fig. 2A). The use of ATP coupled to Sepharose via a spacer of sebacic acid dihydrazide was essential, since commercial adipic acid-ATP or ATP-agarose showed a significantly lower affinity for tubulin S. Westermann and others approx. 100 ng of the enzyme after anion-exchange chromatography (fraction IV), silver-stain. Relative molecular masses ...
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... use of glycerol-gradient centrifugation (Fig. 2B) as the next step had several advantages. First, glycerol stabilised the enzyme. The recovery after this step was typically greater than 70% while simply keeping fraction II at 4°C for 24 hours led to at least 50% loss in activity. Second, the gradient centrifugation provided another tenfold increase in specific activity as most of the ...
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... 38×10 3 for a globular protein. In good agreement with this value, analytical gel filtration of fraction II on Superdex 200 gave a native molecular mass of about 35×10 3 (Rs approx. 28Å; data not shown). However recovery of glutamylase activity in gel filtration was invariably low so that this step could not be used for preparative purification. Fig. 2C shows the subsequent purification of fraction III (pooled fractions from one gradient tube, 2 ml volume) by anion-exchange chromatography on Mono Q. Most of the protein was eluted with 0.25 M NaCl and the activity was released as a sharp peak during the final wash with 0.9 M NaCl. Fraction III enzyme also bound to a cation-exchange ...
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... showed that the final preparation from the Mono Q column is dominated by a single polypeptide with an apparent molecular mass of about 40 kDa (Fig. 2D). Some minor contaminants are still present in this fraction, but their occurrence did not mirror the enzyme activity profile in the column fractions. Because of the presence of high salt the glutamylation activity is strongly inhibited in the final fractions and the salt cannot be efficiently removed by gel filtration or dialysis ...
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... Crithidia tubulin polyglutamylase has a molecular mass of 38×10 3 in glycerol-gradient centrifugation and SDS-PAGE shows that it consists of a single polypeptide chain (Fig. 2). Interestingly tubulin tyrosine ligase, which also catalyses the ATP-dependent synthesis of a peptide bond, is a monomer with a molecular mass of 43×10 3 ( Schröder et al., 1985;Ersfeld et al., 1993). Thus one wonders whether the enzymes responsible for all three tubulin modifications, which involve the ATP- dependent formation of ...
Citations
... Polyglutamylation is an ancient phenomenon evolutionarily conserved from protists to mammalian cells; it is present in sperm flagella of mammals, in sea urchins as well as in several protists including Giardia, Tetrahymena, Crithidia and Trypanosoma (Eddé et al., 1990;Seebeck et al., 1990;Bré et al., 1994;Rüdiger et al., 1995;Moulay et al., 1996;Plessmann and Weber, 1997;Schneider et al., 1997;Weber et al., 1997;Westermann et al., 1999). In mammalian cells, tubulin polyglutamylation is related to centrosome stability, axonemal maintenance and mobility in cilia and flagella, and neurite outgrowth (Gagnon et al., 1996;Bobinnec et al., 1998;Million et al., 1999;Westermann and Weber, 2003;Janke et al., 2005;Ikegami et al., 2006;Pathak et al., 2007;Vogel et al., 2010) (reviewed in Janke et al. (2008). ...
Microtubules are subject to post-translational modifications, which are thought to have crucial roles in the function of complex microtubule-based organelles. Among these, polyglutamylation was relatively recently discovered, and was related to centrosome stability, axonemal maintenance and mobility, and neurite outgrowth. In trypanosomatids, parasitic protozoa where microtubules constitute the essential component of the cytoskeleton, the function of polyglutamylated microtubules is unknown. Here, in order to better understand the role of this conserved but highly divergent post-translational modification, we characterised glutamylation and putative polyglutamylases in these parasites. We showed that microtubules are intensely glutamylated in all stages of the cell cycle, including interphase. Moreover, a cell cycle-dependent gradient of glutamylation was observed along the cell anteroposterior axis, which might be related to active growth of the microtubule ‘corset’ during the cell cycle. We also identified two putative polyglutamylase proteins (among seven analysed here) which appeared to be clearly and directly involved in microtubule polyglutamylation in in vitro activity assays. Paradoxically, in view of the importance of tubulins and of their extensive glutamylation in these organisms, RNA interference-based knockdown of all these proteins had no effect on cell growth, suggesting either functional redundancy or, more likely, subtle roles such as function modulation or interaction with protein partners.
... After that, a second enzyme, a tubulin polyglutamylase, adds a series of glutamates to the first one through α/α linkages (88). A tubulin polyglutamylase has been purified from the trypanosome Crithidia (89). It forms a complex with the tubulin dimer and can add glutamates to both αand β-tubulins from brain (89). ...
... A tubulin polyglutamylase has been purified from the trypanosome Crithidia (89). It forms a complex with the tubulin dimer and can add glutamates to both αand β-tubulins from brain (89). In this respect, the Crithidia enzyme is different from the putative mammalian brain equivalent, which preferentially glutamylates α. ...
The tubulin molecule is unusual because of the number and nature of post-translational modifications that it undergoes. These
modifications may be involved in regulating microtubule stability and interactions with microtubule-associated proteins, but
they also may have as yet undiscovered functions. Certain of these modifications are found in many other proteins; these include
phosphorylation of a serine residue in β-tubulin and acetylation of a lysine residue in α-tubulin. Other modifications occur
exclusively, or almost exclusively in tubulin. Among these are the removal and addition of a tyrosine at the C-terminus of
α and the addition of several glutamate or glycine residues to the γ-carboxyl group of glutamate residues in the C-terminal
regions of both α and β. The identification of the mechanisms by which these modifications occur and of their roles in microtubule
assembly and function are currently very active topics of research; they will be addressed in this chapter.
... In contrast, basal body formation and maturation in S. similis are consecutive events: new basal bodies are rapidly integrated into the fibrous system surrounding the mature basal bodies (centrin fibers, dand s-fibers), achieve an active role in premitotic cells by organizing microtubules at their proximal end [Lechtreck and Grunow, 1999] , and template the assembly of axonemes during cytokinesis. Recently, it has been shown that tubulin-polyglutamylase is associated with the Tri- ton-X-100 insoluble cytoskeleton of trypanosomes [Westermann et al., 1999] and prefers microtubules over tubulin as a substrate [Regnard et al., 1998], supporting the view that polyglutamylation occurs after assembly of a microtubular probasal body. In contrast, GT335 decorated fibrous granules, which are supposed to be basal body precursors during ciliogenesis [Million et al., 1999] and hyperpolyglutamylation of tubulin, as it seems also characteristic for centrioles and basal bodies, occurs in vitro only on soluble tubulin [Regnard et al., 1998]. ...
Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.
... According to that model, the results obtained with HeLa TPG would imply that the same γE is able to both initiate and elongate side chains. Very recently, TPG was isolated from the trypanosomatid Crithidia fasciculata (Westermann et al., 1999). In this case, the enzyme activity was shown to glutamylate preferentially tubulin which is already glutamylated. ...
... The genes encoding the(se) polypeptide(s) are still unidentified. A purified enzyme fraction from C. fasciculata contains a single polypeptide of 40 kDa, perhaps corresponding to a catalytic subunit (Westermann et al., 1999). No sequence information has yet been published for this protein. ...
Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for alpha- or beta-tubulin subunits. In the latter case, the specificity probably also concerns the different beta-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G(2)-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.
Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and β-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in β-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.
The cytoskeleton plays a fundamental role in various processes such as the establishment of cell shape, cell locomotion, and
the intracellular motility of various structures found in eukaryotic cells. Microtubules are among the most conspicuous structures
in the cytoskeleton. They can be found free in the cytoplasm, forming the mitotic spindle or assembled in various structures.
A special type of microtubule arrangement is found in some protozoa, whereby they are organized as a single layer located
immediately below the plasma membrane, constituting what is generally referred to as subpellicular microtubules. This special
array of microtubules is found in members of the Kinetoplastida family and in the Apicomplexa phylum. Here, we review basic
aspects of subpellicular microtubules, emphasizing their visualization as a whole network, their structural organization,
their heterogeneity as analyzed using an immunocytochemical approach, some of their biochemical properties, and their sensitivity
to drugs, as well as their functional role.
Tubulins undergo unique post-translational modifications, such as tyrosination, polyglutamylation, and polyglycylation. These modifications are performed by members of a protein family, the tubulin tyrosine ligase (TTL)-like (TTLL) family, which is characterized by the presence of a highly conserved TTL domain. We and others have recently identified tubulin polyglutamylases in the TTLL family [Janke, C., et al. (2005) Science 308, 1758-1762; Ikegami, K., et al. (2006) J. Biol. Chem. 281, 30707-30716; van Dijk, J., et al. (2007) Mol. Cell 26, 437-448]. Previously, we identified TTLL7 as a beta-tubulin-selective polyglutamylase. However, there is controversy over whether TTLL7 functions as an initiase, elongase, or both in polyglutamylation. In this report, we investigate the polyglutamylation reaction by TTLL7 by employing a recombinant enzyme and in vitro reaction. Two-dimensional electrophoresis and tandem mass spectrometry showed that TTLL7 performed both the initiation and elongation of polyglutamylation on beta-tubulin. Recombinant TTLL7 performed with a maximal and specific activity to polymerized tubulin at a neutral pH and a lower salt concentration. The initial rate and inhibitor analyses revealed that the mechanism of binding of three substrates, glutamate, ATP, and tubulin, to the enzyme was a random sequential pathway. Our findings provide evidence that mammalian TTLL7 performs both initiation and elongation in the polyglutamylation reaction on beta-tubulin through a random sequential pathway.
The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.
Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.
