Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms

The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha-tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.     

Posttranslational tyrosination/detyrosination of tubulin

Tubulin can be posttranslationally modified at the carboxyl terminus of the alpha-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of alpha-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of alpha-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.     

Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55-kDa subunits, alpha- and beta-tubulin; in Trypanosoma brucei, the tubulin-coding sequences are clustered in a 40-kb fragment of tandemly repeated alpha- and beta-tubulin genes separated by a 170-bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5' 35-nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post-translationally modified in 2 ways: the C-terminal tyrosine of alpha-tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin-tyrosine ligase; alpha-tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the beta-tubulin form defined by the monoclonal antibody 1B41 is sequestered into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so-called "subpellicular microtubule quartet" (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.     

Partial purification and characterization of microtubular protein from Trypanosoma brucei

The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.     

Characterization of a major brain tubulin variant which cannot be tyrosinated

Brain tubulin preparations contain an abundant type of tubulin which does not undergo the normal cycle of tyrosination-detyrosination, and whose nature is still unknown. We have used peptide sequence analysis and mass spectrometry combined with immunological procedures to show that this non-tyrosinatable tubulin has a specific primary structure. It differs from the tyrosinated isotype in that it lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit. Thus, non-tyrosinatable tubulin originates from a well-defined posttranslational modification of the tubulin primary structure which is located at the expected site of activity of tubulin tyrosine ligase. This probably accounts for the reason why it cannot be tyrosinated. The significance of this abundant brain isotubulin and the metabolic pathway involved in its formation remain to be elucidated. This should shed light on the relation between the structural diversity of the carboxy terminus of alpha-tubulin and the regulation of functional properties of microtubules.     

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.     

Tubulin tyrosinolated in vivo can be different from that tyrosinolated in vitro

Tubulin can be tyrosinolated, in the presence of ATP, by tubulin-tyrosine ligase, and tyrosine can be released by the same enzyme in the presence of ADP plus inorganic phosphate. There is however a 'non-substrate' component of tubulin which can not be tyrosinolated or detyrosinolated by this enzyme. Tubulin tyrosinolated in vivo was found to be the non-substrate species in HeLa cells, and the substrate species in cultured neuronal cells. In this respect HeLa tubulin resembled membrane-associated tubulin from brain, and neuronal cell tubulin resembled brain cytosolic tubulin.     

Tyrosination of microtubules and non-assembled tubulin in brain slices

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded. We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules. The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.     

Two related subpellicular cytoskeleton-associated proteins in Trypanosoma brucei stabilize microtubules

The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed in T. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis.     

Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine on microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymerized tubulin pools were separated and tubulin was quantified using a competitive ELISA. Carbendazim and colchicine caused extensive microtubule depolymerization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained relatively constant after carbendazim treatment but decreased twofold after colchicine treatment. To determine if modifications to tubulin may be associated with polymerization status, tubulin pools were analyzed by immunoblotting. Acetylated alpha-tubulin and betaIII-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha-tubulin (52 kDa) was localized in both tubulin pools and had decreased tyrosination in the microtubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated alpha-tubulin and general alpha-tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alpha-tubulin isoform) lost tyrosination, yet was still present in the microtubule pool based on detection with the general alpha-tubulin antibody, after carbendazim treatment. Similar effects were seen with colchicine, although loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymerization of microtubules.     

Stabilization of post-translational modification of microtubules during cellular morphogenesis

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Do TNF-alpha-insensitive cancer cells escape alpha-tubulin nitrotyrosination?

TNF-alpha induces tumor-selective cytotoxicity in certain cancers, but many tumors are resistant to the effects of this inflammatory cytokine. This brief hypothesis outlines my views that nitric oxide-mediated alpha-tubulin posttranslational nitrotyrosination may be a major mechanism through which TNF-alpha exerts its cytotoxic effects on cancer cells. Additionally, I propose that tumor cells that are resistant to the effects of TNF-alpha may be so because of suppressed levels of "tubulin tyrosine ligase," which is responsible for the posttranslational tyrosination of alpha-tubulin.     

Identification by mass spectrometry of a new alpha-tubulin isotype expressed in human breast and lung carcinoma cell lines

The extensive C-terminal molecular heterogeneity of alpha- and beta-tubulin is a consequence of multiple isotypes, the products of distinct genes, that undergo several posttranslational modifications. These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha-tubulin, and phosphorylation of the beta III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human cell lines. Total cell extracts are resolved by SDS--PAGE and transferred to nitrocellulose, and the region of the blot corresponding to tubulin (approximately 50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human alpha- and beta-tubulin CNBr-derived C-terminal peptides are all in the 1500--4000 Da mass range and can be analyzed directly by MALDI-TOF mass spectrometry in the negative ion mode without significant interference from other released peptides. In this study, the tubulin isotype diversity in MDA-MB-231, a human breast carcinoma cell line, and A549, a human non-small lung cancer cell line, is reported. The major tubulin isotypes present in both cell lines are k-alpha 1 and beta 1. Importantly, we report a previously unknown alpha isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of alpha-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-alpha 1 and beta 1 isotypes was observed in these two human cell lines.     

Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.

A cycle of posttranslational modification of alpha-tubulin has previously been described in higher eukaryotes, in which a C-terminal tyrosine residue is removed and replaced by two complementary cytoplasmic enzymes. The activity of the detyrosinating enzyme, tubulin carboxypeptidase (TCP), and its potential for regulating the level of detyrosinated (Glu) subunits in microtubules (MTs) is of great interest, since TCP catalyzes the primary modification of tubulin and since the level of Glu alpha-tubulin in MTs increases during a variety of differentiative and morphogenetic events. As a first step in examining the role of TCP in cellular morphogenesis, it was necessary to develop an assay for TCP with sufficient sensitivity and specificity to detect TCP activity during these events. Unlike previously described assays for TCP, ours makes use of the affinity TCP exhibits for MTs. NGF-induced neurite outgrowth in PC-12 cells was accompanied by a moderate (approximately 2-fold) increase in TCP activity, while myogenesis of L6 cells resulted in an almost insignificant decrease in activity. Measurements of TCP activity during differentiation were correlated with the level of extract Tyr tubulin, which increased (approximately 37%) during neurite outgrowth and was unchanged during myogenic differentiation. Our results suggest that TCP activity is regulated relative to its substrate, Tyr tubulin, and that changes in MT dynamics, rather than enzymatic activities, are the primary determinants of MT posttranslational modification state during differentiation. In addition, the assay we have devised for TCP and the characterization of TCP during differentiation may allow the future delineation of the mechanism(s) of regulation of TCP and the role this enzyme plays in modulating MT function during differentiation.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.