Tubulin Post-Translational Modifications - Wiley Online Library

Eur. J. Biochem. 244, 265-278 (1997) 0 FEBS 1997

Review nbulin post-translational modifications Enzymes and their mechanisms of action Thomas H. MacRAE Department of Biology, Dalhousie University, Halifax, Canada (Received 13 September 1996) - EJB 96 1366/0

This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosinationltyrosination, acetylationldeacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable a-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and a-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulinlmicrotubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications. Keywords: tubulin; microtubule ; post-translational modification ; tyrosination ; detyrosination ; non-tyrosinatable ; acetylation; phosphorylation; polyglutamylation ; polyglycylation.

Of the three major isotypes of tubulin in eukaryotic cells, atubulin and P-tubulin form heterodimeric complexes which associate head-to-tail into protofilaments and then laterally to make up the wall of the cylindrical microtubule [I-31. The third isotype, y-tubulin, appears in the cytosol and in microtubule-organizing centers as ring-shaped structures where it nucleates microtubules [4-61. Both a-tubulin and P-tubulin, and perhaps y-tubulin [7], exist within most eukaryotic cells as families of closely related isoforms. Isotubulin composition varies spatially and temporally, determined by differential transcription of a-tubulin and P-tubulin genes within small families [2, 8, 91 and by post-translational modifications of tubulin [I, 8, lo]. Several types of post-translational modifications affect tubulin and some may be restricted to this protein. All of the modifications described in this review are enzymatically driven, providing the potential for rapid changes to tubulin, most are reversCorrespondence to T. H. MacRae, Department of Biology, Dalhousie University, Halifax, Nova Sdotia B3H 451, Canada

F a : i - 1 902 494 3736. Abbreviations. MAP, microtubule-associated protein ; EGF, epidermal growth factor; NCAM, neural-cell-adhesion molecule. Note. This Review will be reprinted in EJB Reviews 1997 which will be available in April 1998.

ible, and all but acetylation occur at the highly variable carboxy terminus of tubulin [ll]. None of the changes is, however, essential to survival, because in cases where extensive analyses were done there are examples of cells which lack the post-translationally generated isoform under consideration [ 12- 141. One consequence of tubulin post-translational modification is to mold the isoform composition of the microtubule wall. The chemical heterogeneity of the microtubule surface is increased, either because there is a greater selection of isofoms for assembly or the tubulin is modified once it polymerizes. Diversity may also decrease if the same chemical group is added to or removed from all or most tubulins within a cell. This is important if a predominant modification masks a reactive site on tubulin and limits its interaction with other cellular components. Additionally, microtubules are divided into discrete regions if the posttranslational change is restricted to or occurs more rapidly in one area of the polymer. Segregation of isoforms has the potential to reveal the growth profile of individual microtubules and/or provide, as in axons, stable nucleating structures for the positioning of microtubules [15, 161. Whatever the case, post-translational changes, in the absence of differential gene expression, generate diversity within microtubule populations, possibly influencing their functions. For this reason, tubulin post-translational modifi-

266

MacRae ( E m J. Biochem. 244)

cations have been studied extensively. One aspect of the work concentrates on the enzymes that modify tubulin and they are the focus of this review.

Qrosinated, detyrosinated and non-tyrosinatable tubulins Most a-tubulins, but not all [9, 171, contain a gene-encoded carboxylterminal tyrosine. The tyrosine is removed to give detyrosinated tubulin, characterized by a terminal glutamic acid, and subsequently reattached to again form tyrosinated tubulin [8, 10, 181. Detyrosinated tubulin is found in stable microtubules but the change is a consequence rather than the cause of stabilization [13, 19, 201 and stable microtubules do not necessarily have detyrosinated tubulin [21]. Removal of both tyrosine and glutamic acid yields non-tyrosinatable tubulin, perhaps the major a-tubulin in mammalian brain [22, 231. The non-tyrosinatable isoform is usually a component of very stable microtubules and it may no longer participate in the detyrosinatiodtyrosination cycle.

Tubulin-specificcarboxypeptidase The primary event in the detyrosination/tyrosination cycle is removal of the carboxy-terminal tyrosine of a-tubulin, mediated by tubulin-specific carboxypeptidase in a process proposed to occur mainly, if not exclusively, on microtubules [13, 18, 24261. The carboxypeptidase is thought to bind microtubules and to prefer polymerized over dimeric tubulin as substrate. Evidence supportive of these proposals includes the delayed appearance of detyrosinated, as opposed to tyrosinated tubulin in cellular microtubules regrown after disassembly by tubulin-reactive drugs, exposure to cold and the onset of mitosis 113, 24, 251. Moreover, enhanced detyrosination of tubulin in microtubules stabilized by taxol or by other means suggests that detyrosination happens preferentially on microtubules [13, 24, 271. Although these data indicate that tubulin detyrosination occurs after its incorporation into microtubules, they do not directly address the question of carboxypeptidase specificty. That is, does this enzyme favor polymerized tubulin as substrate or does it compete poorly with tubulin tyrosine-ligase for tubulin dimers ? The latter enzyme adds tyrosine to soluble, detyrosinated tubulin and apparently does not react efficiently with tubulin once it is incorporated into microtubules (described below). Thus, it can be argued that taxol-stabilized microtubules within cells are detyrosinated because all of the tubulin is in microtubules and not accessible to the ligase. Moreover, the pool of non-assembled tubulin is tyrosinated because the ligase acts more quickly upon the tubulin dimer than does the carboxypeptidase, even through the latter enzyme acts equally well with either polymerized or soluble tubulin. In this context, Wehland and Weber [13] have proposed that tubulin carboxypeptidase is the rate limiting enzyme in the cycling of tyrosine. To test such possibilities an interesting variation on the analysis of carboxypeptidase activity in vivo is to depolymerize microtubules by drug treatment and then inject cells with an antibody that inhibits tubulin tyrosineligase [13]. Under these conditions soluble tubulin is not detyrosinated, in spite of the presence of carboxypeptidase in cells. The experiments reinforce the conclusion that tubulin carboxypeptidase prefers polymerized substrate and that it binds to microtubules as a prerequisite of function. Tubulin carboxypeptidase has not been purified to homogeneity, nor has its gene been cloned and sequenced, information required for a full understanding of the enzyme. Partial purifications of putative tubulin-specific carboxypeptidases by standard biochemical procedures are available; in one case the enzyme obtained was unstable upon storage [28-301 and in another it

was stable for months [31]. The interaction of these enzymes with proteins other than tubulin was examined, yielding conflicting reports that aldolase serves as a substrate [28, 311. The discrepancies perhaps rest in the use of different molar ratios of aldolase to enzyme. Additionally, the assays of carboxypeptidase activity were performed in complex mixtures of proteins rather than in well defined systems with purified components. Soluble tubulin was often employed in the assay systems [28, 30, 32, 331, a suitable approach at the time because both assembled and non-assembled tubulin were thought to be carboxypeptidase substrates [34-361. There were suggestions throughout much of this earlier work, however, that the tubulin carboxypeptidase utilized polymerized tubulin more effectively. A particularly compelling experiment was done by Arce and Barra [371 wherein the use of tubulin-tyrosine complexed to colchicine indicated that detyrosination depended on tubulin polymerization, or at least that detyrosination was greater under these conditions. A tubulin-reactive carboxypeptidase was recently partially purified and assayed on the basis of its affinity for microtubules [38, 391. It was concluded that microtubule dynamics are more important than enzyme activities in determining the extent of tubulin detyrosination during differentiation of neural and muscle cells. Previously, researchers [32, 401 had indicated that tubulin detyrosination is influenced by factors other than the amount of carboxypeptidase, but their assays did not employ taxol-stabilized microtubules. The carboxypeptidase described by Webster et al. [38], which undergoes a 60-80% decrease in activity upon reduction in pH to 5.4, appears to modify polymerized, as opposed to soluble tubulin, although substrates were not compared under identical conditions. Rather than mixing soluble tubulin with cell-free extracts, it would have been preferable to strip the carboxypeptidase from microtubules, this appears to be possible [39], and test the enzyme so obtained with soluble tubulin under conditions the same as those employed in assays where microtubules were used. A tubulin-reactive carboxypeptidase has been partially purified from Artemia and shown to be more active with soluble tubulin than taxol-stabilized microtubules, but its role in tubulin modification in vivo is uncertain 1411. A tubulin-reactive carboxypeptidase from rat brain has affinity for microtubules [37, 40, 421 and centrifugation of samples to remove microtubules depletes carboxypeptidase activity in the resulting supernatants [37]. Erythrocytes from chicken and toads, however, were reported to contain soluble carboxypeptidase [33], but the activities of the enzymes were not determined on stabilized microtubules. In a related vein, tubulin-reactive carboxypeptidases from brain detyrosinates non-native tubulin [30, 431, a result similar to that obtained with carboxypeptidase A, and the tubulin carboxypeptidase prepared by Kumar and Flavin [31] utilizes vinblastine-induced oligomers. Such results are important when analyzing putative tubulin-specific carboxypeptidases because evidence based on inhibitor studies shows that the condition of tubulin employed in assays is critical [38] ; for example, soluble tubulin is inappropriate because other carboxypeptidases in test preparations confound the results. The specificity of the carboxypeptidases and the preference for polymer versus dimer would therefore appear to be uncertain in several studies because the enzymes were not assayed on stabilized microtubules, even though preparation may have involved a step in which they were bound to microtubules [34, 37, 40, 421. Additionally, specific activities for enzymes in supernatants recovered after pelleting microtubules are missing, and the amount of enzyme sticking to microtubules relative to that initially present in cell-free extracts cannot be determined. In other work, the preference for polymerized tubulin of a carboxypeptidase, which functioned better at pH 6.8 than at 5.5

MacRae ( E m J. Biochem. 244)

[31] and was relatively insensitive to 1,lO-phenanthroline, an inhibitor of carboxypeptidase A [44], was shown clearly. However, the enzymes were isolated from brain supernatants prepared by assembling the tubulin and discarding the microtubules. Although there may be variation in tubulin carboxypeptidases from one cell type to another, and the experimental conditions are not identical, Webster et al. [38] indicate that tubulinspecific carboxypeptidase is lacking in post-polymerization supernatants and that enzymes therein are not responsible for tubulin detyrosination in vivo. This possibility arises elsewhere [37, 40, 421, keeping in mind that the microtubule associated enzymes in these studies are assayed on soluble tubulin or on microtubules under apparent steady-state conditions, not on stabilized microtubules.

Tubulin tyrosine-ligase Tubulin tyrosine-ligase, the enzyme that reattaches tyrosine to detyrosinated tubulin was detected in rat brain cell-free preparations which incorporated [14C]tyrosine[45 -471. Labelling of proteins with tyrosine requires ATP, Mg2+and K', and is RNase insensitive, demonstrating that amino acid addition is not the consequence of protein synthesis. The tyrosinating activity is enriched in brain, developmentally regulated, declines with increasing age of animals and is lost upon storage at 4°C. Phenylalanine and L-Dopa (~-3,4-dihydroxyphenylalanine)are added to brain proteins in vitro under the same conditions as tyrosine [46, 471. Moreover, either phenylalanine or L-Dopa inhibits the incorporation of tyrosine into a position, suggested by Barra et al. [47], through use of carboxypeptidase A, to be the carboxy terminus of the accepting protein. The inhibition, competitive in nature, is reciprocal because tyrosine prevents the use of both phenylalanine and L-Dopa. Chromatographic behaviour of labelled proteins, migration during SDS/polyacrylamide gel electrophoresis and precipitation with vinblastine sulphate are early indications that tyrosine is added specifically to tubulin in brain cell-free extracts [48]. This proposal was confirmed by purification and/or vinblastine sulphate precipitation of radioactively labelled protein from brain, followed by SDS polyacrylamide gel electrophoresis, showing all radioactivity in the a-subunit of tubulin [49, 501. The incorporation of either L-Dopa or phenylalanine is identical to that of tyrosine, and it was estimated that 30% of rat brain tubulin is modified by addition of these compounds [49]. Subsequently, atubulin was shown to be tyrosinated in vivo and in vitro, either by attachment of [14C]tyrosineto a carboxypeptidase-A-sensitive position in the presence of cycloheximide to inhibit protein synthesis [51], or by demonstrating the enhanced incorporation of tyrosine into tubulin after its treatment with carboxypeptidase A [W. During initial characterization an unstable tyrosine-ligase activity was separated from tubulin and a 5-20-fold purification achieved [SO, 521. The enzyme has an apparent molecular mass of 150kDa when in cell-free extracts but this is reduced to 35 kDa after anion-exchange chromatography, a size difference apparently based on association of the ligase with tubulin [52]. The ligase removes tyrosine from tubulin when ATP is replaced by ADP and P, [50, 521. Also, 6 s tubulin dimers, but not stable axonemal microtubules from Tetrahymena, are excellent substrates, one of the first suggestions that the ligase prefers soluble over polymerized tubulin. It is difficult, however, to exclude the possibility that accessory proteins shielded enzyme-reactive sites on the axonemal tubulin. Raybin and Flavin [50,53] showed that tubulin polymerization is not inhibited by addition or removal of tyrosine at its carboxy terminus, a finding noted by others [34], and all rat tissues they examined exhibit significant ligase activ-

267

ity [53]. However, tubulin tyrosine-ligase was not found at this time in Tetruhymena and yeast, and only a low level, at best, was observed in sea urchins [53]. Others reported the absence of tubulin tyrosine-ligase from invertebrate tissues [54] but it was eventually shown that if the enzyme is partially purified it can be detected easily in invertebrates, even though it still was not seen in Teti-ahymena [55]. Tubulin tyrosine-ligase is now known to enjoy a wide phylogenetic distribution. Schroder et al. [56] prepared tubulin tyrosine-ligase by standard biochemical methods and immunoaffinity chromatography, obtaining enzyme preparations that are greater than 95 % pure and achieving good recovery. The enzyme, when purified either to a low level [50,521 or to a much higher degree [57],requires glycerol for stability [56] overcoming problems occasioned by ready loss of activity. The purified tyrosine-ligase is a monomeric protein with a molecular mass of about 40 kDa and it forms a 1 :1 complex with tubulin [52, 561, verifying previous work on the protein. The ligase has a recognition site on ptubulin and interaction with a/l-tubulin is not perturbed by removal of its carboxy-terminal region with subtilisin, or by YL1/2, a monoclonal antibody that attaches to the carboxy end of tyrosinated a-tubulin [58]. The specificity of the ligase for tubulin may in part reside with the contribution made to the recognition site by the p-subunit. Additionally, if this region is hidden when assembly occurs, the preference of the purified enzyme for soluble tubulin is explained [%]. Similar substrate specificity of the ligase is indicated in other studies [34, 50, 591, as is the inefficient binding of ligase to microtubules [52, 601. These results are somewhat difficult to interpret due to dynamic instability of the microtubules used in the assays and/or the presence of carboxypeptidases in the cell-free systems under study. To further investigate tubulin tyrosine-ligase specificity, interaction of the enzyme with synthetic carboxy-terminal peptides of a-tubulin was tested [61]. The carboxy-terminal tetradecapeptide of detyrosinated a-tubulin serves as substrate, however at a 50-fold lower efficiency. Modifying single amino acid residues and varying its length reveals maximal tyrosination once a peptide ending in Gly-Glu-Glu reaches 12 residues. Tyrosine is only added to a peptide terminating in glutamic acid and this residue is favored over all others in the adjacent position, but aspartic acid will work. The requirement for glycine in the third position following the two glutamic acids is strong, although serine for example, permits 30% activity. Peptides ending in Gly-Glu fail to function as substrates and the reactivity of tubulin tyrosineligase is unaffected by polyglutamylation (described below). The gene for porcine tubulin tyrosine-ligase has been cloned and sequenced [62]. Its primary transcript is an mRNA of approximately 6 kb, yielding a protein of 379 amino acid residues with a molecular mass of 43.4 kDa and a PI of 6.51. No other protein with extensive sequence similarity is known. A putative serine-phosphorylation motif (residues 73 -76) exists, and residues 244-258, exposed on the exterior of the protein, contain a site for V8-protease action. This enzyme nicks tubulin tyrosineligase, producing closely associated fragments of 14 kDa and 30 kDa that are able to complex with tubulin [58]. The ATP and P-tubulin-binding domains reside on the 30-kDa portion of the ligase while the 14-kDa fragment possibly harbours part of the catalytic region. The analysis does not reveal the exact location where ATP binds to the 30-kDa fragment, but it shows that a P-loop sequence, characteristic of many ATP-binding regions, is absent. Three glycines are located within residues 154- 159, and this sequence may consitute the ATP-binding region. Knowing the tubulin tyrosine-ligase sequence allows its manipulation, providing a way to understand the physiological function of tyrosination-detyrosination. Ersfeld et al. [62] propose the use of antisense oligonucleotides or mRNA to inhibit the enzyme for

268

MacRae (Eul: J. Biochem. 244)

translational modification is independent of protein synthesis but requires flagellar growth, that the responsible enzyme is stored in the cell body and activated upon transfer into the axonemal matrix [72, 761. An acetyltransferase was detected in Chlamydomonas flagella and it functioned specifically with a-tubulin from Chlamydomonas and mammalian brain, yielding 1.1 mol aceNon-tyrosinatable tubulin tate/mol substrate [77, 781. Characterization of tyrosinated and detyrosinated tubulin reAs a prerequisite to understanding its function, a-tubulin vealed an isoform which is not tyrosinated [53, 63, 641. This acetyltransferase was purified from Chlamydomonas [79]. A non-tyrosinatable a-tubulin, purified by affinity chromatography 110-fold enrichment was achieved and the preparation contains [65], is lacking both tyrosine and glutamic acid from its carboxy a major polypeptide of 67 kDa and other minor proteins. If the terminus [66]. The absence of glutamic acid explains why tu- 67-kDa protein is a-tubulin acetyltransferase, then the enzyme bulin tyrosine-ligase, with a minimal recognition sequence of is probably a homodimer because it elutes from Sephadex GGly-Glu-Glu, cannot reattach tryosine [61]. The isoform, termed 150 at the volume expected for a 130-kDa protein. Tubulin aceA2-tubulin [22], is enriched in neuronal tissue, accounting for tyltransferase was obtained from mammalian brain by Greer et at least 35% of total brain tubulin [22, 651, but it is essentially al. [77]who separated the enzyme from tubulin by chromatogranon-existent in rat muscle [23]. In cultures of fibroblasts and phy on phosphocellulose. The presence of a-tubulin acetylbovine adrenal cortical cells, d2-tubulin is localized mostly in transferase in neural tissue has been confirmed [go]; the enzyme primary cilia and centrioles, structures with very stable microtu- purifies with tubulin upon temperature-dependent cycling of bules [22], and it is found in the axonenial tubulin of sperm from microtubule proteins and it probably acetylates microtubulesea urchins [67]. Somewhat unexpectedly, d2-tubulin resides in associated protein (MAP2). Sequencing of peptides obtained growth cones, transient structures lacking detyrosinated tubulin, by reverse-phase HPLC after protease digestion reveals that itself an indicator of durable microtubules. In spite of this latter [3H]acetylated a-tubulins from mouse brain and Chlamydomoobservation, A2-tubulin normally occurs in long-lived microtu- nas are labelled at the same lysine residue by neural a-tubulin bules and it is probably a useful marker for such polymers [22]. acetyltransferase. More recently, Lloyd et al. [81] partially puriExcluding the unlikely presence of a gene for A2-tubulin, fied a-tubulin acetyltransferase from bovine brain. The most stabilization of cellular microtubules with taxol or vinblastine, prominent polypeptide in their preparation has a molecular mass followed by immunofluorescent staining, suggests that d2-tu- of 62 kDa, a value close to that for the acetyltransferase from bulin is generated by long exposure to a modifying enzymes(s) Chlamydomonas. Additionally, these workers observed an a-tu[22]. Either a carboxypeptidase(s) sequentially removes tyrosine bulin acetyltransferase in extracts of bovine retinal tissue and it and glutamic acid from the carboxy terminus of a-tubulin, or a may be very similar to the enzyme from Chlamydomonas fladipeptidase cleaves both amino acids simultaneously [22, 661. gella [81]. There is little evidence in favour of one mechanism over anDuring purification of a-tubulin acetyltransferase from Chlaother, but the presence of tyrosinated and A2-tubulin in growth mydomonas evidence was obtained for a tubulin deacetylase and cones, in the absence of detyrosinated tubulin, supports the sec- an inhibitor, perhaps a protein, of a-tubulin acetyltransferase ond option [22]. Recent progress in the characterization of A2- [79], the latter also present in mouse brain [go]. The inhibitors tubulin through application of biochemical and molecular ap- and their role in regulation of a-tubulin acetylation are poorly proaches suggests this question will soon be resolved. defined, while the deacetylase probably removes the acetyl group from a-tubulin upon resorption of Chlamydomonas flagella. Restriction of tubulin deacetylase to the cell body unTubulin acetylation doubtedly explains the low level of acetylated tubulin seen in a-tubulin acetyltransferase and deacetylase. The first indi- this region of Chlamydomonas when 6-1lB-1, a monoclonal ancation of acetylated a-tubulin, a modified protein thought to tibody that specifically recognizes this isoform, is used [82]. have arisen early in eukaryotic cell evolution [68], was obtained Tubulin acetyltransferase has a tight Mg2+-dependentassociwhen a flagellar tubulin from Polytomella was shown to migrate ation with Chlamydomonas axonemal microtubules but modifito a more acidic position on two-dimensional gels than did the cation of a-tubulin does not require Mg" and is inhibited by major cytoplasmic isoform from this organism [69]. The reason Ca2+,likely through an effect on tubulin [79]. The enzyme exfor the difference is not immediately apparent but translation of hibits a 2X preference for polymerized versus soluble tubulin, mRNA from deflagellated cells, both in vitro and in vivo, indi- although in this work stabilization of microtubules with 20 pM cates a post-translational change [70].The same conclusion was taxol inhibits tubulin acetylation [79]. In contrast, microtubules made when Chlamydomonas tubulin was examined [70,71J , and in HeLa and 3T3 cells treated with taxol at 10 pM are reversibly further strengthened because the change occurs even if protein modified, and acetylated tubulin is restricted to microtubules, synthesis is inhibited [70, 721. Post-translational transformation indicating that this change occurs mainly post-polymerization of cytoplasmic into tlagellar a-tubulin depends on flagella as- [83]. Because acetylation can happen very quickly, as in mouse sembly and when these organelles are resorbed, the acidic iso- oocytes [84], acetylated tubulin does not necessarily demarcate form disappears [73], suggesting reversal of the modification by old microtubules. a second enzyme. Tubulin in flagellar microtubules of ChlamyAcetylated microtubules commonly resist drug-induced but domonas is also labelled in the absence of protein synthesis not cold-induced disassembly, although in some cells a portion when cells are incubated with tritiated acetate and there is no of acetylated microtubules is cold resistant [84-861. For examincorporation of label into other flagellar proteins [72]. Based ple, kinetochore microtubules remaining after drug-induced or on these results it was suggested that flagellar tubulin is ace- cold-induced disassembly are acetylated more extensively than tylated. This proposal was verified by chemical analysis of tu- are microtubules before disassembly [84]. It is also possible, bulin from cells incubated in growth medium containing tritiated such as during the early cleavages of the Drosophila syncytial acetate, revealing that the acetyl group is attached to the &-amino blastoderm, that rapid disassembly of microtubules prevents group of an a-tubulin lysine [74] subsequently demonstrated to their acetylation, at least to levels detectable by immunofluoresbe amino acid residue 40 [75].It was reasoned, because the post- cent staining [87]. In this study acetylated microtubules appear

intervals longer than those possible by injection of antibody, while another approach is to disrupt the ligase gene. Similar experiments are possible for tubulin-specific carboxypeptidase once its sequence is determined.

MacRae ( E m J. Biochem. 244)

either as Drosophila embryos develop or if syncytial blastoderms encounter taxol and anoxia, events thought to stabilize microtubules. Microtubule stability is not, however, the only factor to determine the extent of tubulin acetylation. The mitotic spindle microtubules of Trichomonas vaginalis [68] and of early Xenopus embryos [88] are acetylated when cells are undergoing rapid cleavages. As division slows, acetylated tubulin disappears from Xenopus embryos, suggesting the acetyltransferase is inactivated or removed. How the intracellular spatial organization of acetylated microtubules is determined is an important unanswered question. Upon disassembly the distribution of acetylated microtubules may change in relation to the entire population of microtubules because the quantity of tubulin acetyltransferase is constant in the face of decreasing substrate. Thus, a higher proportion of microtubules is acetylated and their distribution is more widespread within the cell. However, newly formed microtubules may be acetylated almost immediately, as for microtubules in the mouse upon recovery from cold-induced disassembly, but their distribution changes as substrate increases without a corresponding rise in tubulin acetyltransferase. These proposals indicate why the spatial organization of acetylated microtubules varies, but do not explain the compartmentalization of acetylated tubulin in stabilized microtubules. That is, not only are certain microtubules modified, but acetylated tubulin is often in discrete polymer domains tending to turn over more slowly than other regions [891. The results reflect tubulin acetyltransferase function and posit, reinforced by the coorelation between acetylation and increased stability [78, 86, 87, 901, that binding of the enzyme to microtubules is regulated [89]. There may be a factor(s) limiting enzyme activity to certain cellular microtubules and to restricted regions of these structures. One candidate is a MAP which either enhances or inhibits acetyltransferase interaction with microtubules. Another possiblility is that the interplay of microtubules with cytoskeletal elements or intracellular organelles generates a platform for enzyme activity. Such speculations are consistent with known properties of the acetyltransferase and of microtubules. Just what is the role of acetylated microtubules and how does tubulin acetyltransferase contribute to cell function? Acetylated tubulin is not required for survival because it is missing from PtK, cells [83] turkey erythrocytes [91] and Plasmodium falciparum [92], even though lysine may be present at site 40. Two attempts have been made to determine how elimination of Lys40, and thus tubulin acetylation, affects cells. In one study Chlamydomonas is transformed with an a-tubulin gene whose product is not acetylated because Lys40 is replaced by another amino acid [93]. Although the mutated protein constitutes 70% of the flagellar a-tubulin in some transformants, there is no observable phenotype, suggesting that acetylated tubulin has a very subtle function in Chlamydomonas not detectable under laboratory growth conditions. However, as discussed by the authors [93], the threshold level of mutated tubulin may not have been reached, leaving sufficient wild-type tubulin for normal cell function. In a second study, a-tubulin in Tetrahymena thermophila with lysine at position 40 is completely replaced by tubulin containing arginine [94]. These mutants have no acetylated atubulin and are indistinguishable from wild-type cells, indicating that acetylation of Lys40 is not required. But, as noted by Kozminski et al. 1931, replacement with another amino acid demonstrates that lysine is non-essential; it fails to prove that acetylation is dispensable if lysine is present. A different idea is that acetylation of lysine in tubulin is a secondary effect of the acetyltransferase and its primary role is to modify another protein [94]. Clearly, a critical experiment is to prevent expression of the tubulin acetyltransferase and determine the effect this has on

269

a cell which normally acetylates Lys40 in its a-tubulin. Demonstrating that the 62-67-kDa protein mentioned earlier is indeed tubulin acetyltransferase, and acquiring sufficient protein sequence to allow cloning of its gene, is a feasible approach to eliminate the enzyme by genetic manipulation and achieve a greater appreciation of tubulin acetylation.

Tubulin phosphorylation Cyclic-AMP-dependent tubulin kinases. Goodman et al. [95] demonstrated, in work designed to determine the relationship between CAMP,microtubules and secretion, that CAMP enhances phosphorylation of ‘neurotubule subunits’ from bovine cerebral cortex on serine residues. Subsequently, Eipper [96, 971 found that rat brain tubulin incubated in buffer containing radioactive orthophosphate is only modified on a serine residue of the P-subunit. Tubulin was also labelled in vivo and the modified serine is restricted to one very acidic tryptic peptide [97], probably corresponding to the carboxy terminus of the protein. Additionally, Eipper showed that reaction of rat brain tubulin in vitro with an endogenous CAMP-independent kinase results in preferential labelling of P-tubulin at 0.1 mol lzP/mol dimer [98]. This tubulin is aggregated but it is not certain whether aggregation or phosphorylation occurs first, nor is it clear exactly what is meant by aggregation. Tryptic maps of tubulin from in vitro preparations exhibit five major phosphorylated peptides, none of which corresponds to the peptide from tubulin labelled in vivo. Thus, early in the study of tubulin phosphorylation the physiological relevance of results obtained in in vitro experiments was questioned [98]. At about the same time as Eipper’s investigations, tubulin from Tetrahymena cilia [99], muscles and brains of chicks, and HeLa cells [loo] was shown to be phosphorylated. In these reports, the kinases, with molecular masses of 53-54 kDa, are insensitive to CAMP, and both enzymes phosphorylate several different proteins. Tubulin from HeLa cells and chick is modified in vivo on serine residue(s) and P-tubulin appears to be a better substrate than a-tubulin [ 1001. Other experiments revealed the phosphorylation of tubulin and MAPS by endogenous CAMP-dependent kinases 1101, 1021. The kinases purify stoichiometrically with tubulin through four polymerization cycles and one of the enzymes, able to phosphorylate dimeric and 30s tubulin, is proposed to have a molecular mass of 280 kDa [IOI]. In the other case [102], MAP1 and MAP2 are phosphorylated more readily in vitro than is tubulin, a protein phosphorylated at a very low level in vivo. Incubation of guinea pig cerebral cortex slices with ’*P yields tubulin, after purification by polymerizatioddepolymerization, labelled in a-subunits and P-subunits with about two times more phosphate in the latter [103]. Approximately 67 % of the radioactivity from slices migrates near the origin of the gel, and is not in tubulin, while under in vitro labelling conditions about 80% of the 32Pis incorporated equally into a-tubulin and P-tubulin. The kinase from guinea pig brain cycles with tubulin through assembly/disassembly and its activity is stimulated by CAMP. Phosphorylation of both tubulin subunits contrasts the results of Eipper [96, 971 and of Forgul and Dahl 11041 who found that labelling is restricted to a subpopulation of P-tubulin isoforms. In these studies tubulin was not purified by assembly/disassembly procedures after brain homogenization, but it was when a-tubulin and /I-tubulin are labelled.

Ca2+/calmodulin-stimulatedtubulin kinases. Burke and DeLorenzo [1051 describe the endogenous phosphorylation of atubulin and P-tubulin by an unstable Caz+/calmodulin-stimulated kinase from rats of interest at this time because Caz+ had been shown to affect tubulin. The kinase fails to purify with tubulin

270

MacRae (Eur. J. Biochern. 2441

during assembly/disassembly and is located in presynaptic nerve threonine and serine in vitro in acidic sequences of tubulin such terminal fractions where it phosphorylates tubulin in a reaction as those found at its carboxy terminus, and the enzyme appears blocked by trifluoperazine, an inhibitor of calmodulin activity to function better with microtubules as substrate [118]. Tubulin [ 1061. Modification of tubulin is thought to influence synaptic phosphorylated by casein kinase I1 is assembly competent [ 118, vesicle function and neurosecretion because release of neuro- 1191. Peptide mapping indicates that tubulin modified in vitro by transmitter from intact synaptosomes correlates with depolarization-dependent phosphorylation of this protein [1071. Tubulin in casein kinase I1 is more similar to tubulin in N2A neuroblastoma synaptic cytoplasm is also a substrate for a Ca2+/calmodulin- cells, than is tubulin subjected to Ca2+/calmodulin-dependentkidependent kinase, much of which associates tightly with a-tu- nase [ 1201. However, caution is necessary because preparation bulin and /?-tubdin [IOS, 1091. This kinase is a major protein in of N2A tubulin includes its assembly, possibly allowing phosisolated postsynaptic densities [110] and it interacts with cold- phorylation after the cells are homogenized. Additionally in this stable microtubules, causing its loss as tubulin is cycled [ l l l , study, low levels of casein kinase I1 with a molecular mass of 1121. The purified kinase is a multimer of approximately 38-40 kDa were shown to reside in preparations of microtubule 600 kDa. It consists of two related subunits of 52 kDa and proteins from pig brain after two cycles of assemblyldisassembly 63 kDa, termed Q and 0,respectively, both with the ability to [120]. The small amount of enzyme recovered is probably due bind calmodulin and autophosphorylate. The enzyme modifies to its loss through binding to cold-stable microtubules, as rep-tubulin residues, with 60% of the label in threonine and 40% ported for Ca2’/calmoddin-dependent kinase [I 111. Immunoin serine, reaching about 0.8 mol phosphate/mol substrate. Phos- fluorescent staining of cultured cells suggests an association bephorylation of other proteins, including a-tubulin and MAP2, is tween the kinase and microtubules, including those in mitotic mostly on serine residues, with a limited amount on threonine spindles and neurites. Similarly, Risnik et al. [I211 described pig and none on tyrosine. It was proposed that Ca2+exerts its effect brain casein kinase I and I1 which copolymerizes with tubulin, on tubulin by activating the kinase, probably a member of the adheres to microtubules and phosphorylates tubulin and MAP2 type I1 family of calmodulin-dependent kinases [ 11I], leading when supplied with [U-’~P]ATP.However, at least one report provides evidence that casein kinase I1 fails to copolymerize to phosphorylation of tubulin and MAPs [103]. Fukunaga et al. [I 131 purified to apparent homogeneity a with tubulin, although it phosphorylates p-tubulin [ 1221. Instead, high-molecular-mass rat brain Ca”/calmodulin-dependent ki- it was proposed that a MAP, a casein-kinase-like kinase, phosnase composed of 11-14 identical monomers, each of 49 kDa. phorylates tubulin. In support of this, tau and p-tubulin are phosThe enzyme phosphorylates a-tubulin and P-tubulin in vitro, al- phorylated at serine and threonine by a 32-kDa kinase purified though p-tubulin is favoured [ 1141, as well as myosin light chain as a MAP [123]. Summarizing these results, Serrano et al. [118] kinase, casein, MAP2 and tau. Phosphorylation occurs on sev- hypothesize that phosphorylation of tubulin by Ca2+/calmodulineral threonines and serines, reducing tubulin polymerization in dependent kinase and casein kinase I1 serves different functions. vitro in the presence of MAP2 and tau. There is a greater impact Specifically, casein kinase I1 modulates the stability of microtuon the rate than the extent of assembly [114]. Additionally, a bules during neurite outgrowth while Ca2+/calmodulin-depenCa*+/calmodulin-dependent kinase from pig brain modifies u- dent kinase produces assembly-incompetent tubulin and influtubulin and p-tubulin and MAP2 [115]. The kinase is con- ences the interaction of tubulin with cell membranes. structed from a major (50 kDa) and a minor (60 kDa) polypepTubulin phosphorylation by tyrosine kinases. The insulintide, with the latter subject to autophosphorylation. Up to 2.6 rnol of phosphate are added to the carboxy end of each mole of receptor kinase from rat placental membrane interacts with poralp-tubulin, with 70% of the incorporation on serine and 30% cine brain tubulin depleted of MAPs, utilizing [Y-’~P]ATPand on threonine, but none on tyrosine. The phosphorylated tubulin incorporating 0.35 mol of phosphate/mol protein 11241. Insulin neither assembles nor interacts with MAPs, perhaps due to a stimulates the reaction about ninefold. Both a-subunits and pconformational change, but treatment with phosphatase yields subunits of unassembled tubulin are substrates, regardless of the assembly-competent tubulin. Another consequence of phosphor- presence of insulin, and tyrosine residues are targeted. Subseylation within the final 4 kDa of the tubulin carboxy terminus is quent polymerization gives microtubules composed of tubulin reversible enhancement of its interaction with artificial mem- subunits equally phosphorylated in their amino and carboxy dobranes constructed from phosphatidylcholine, a neutral phospho- mains, while the remaining soluble tubulin is modified only on lipid [116]. Again, this is thought to arise from a structural its carboxy-terminal tyrosine, indicating that phosphorylation of this residue prevents assembly. However, when microtubules are change in tubulin. phosphorylated, p-tubulin is preferred and tyrosine on the carPhosphorylation of tubulin by casein kinase I and 11. Tu- boxy terminus of a-tubulin is not changed. The insulin-receptor bulin and MAPs are phosphorylated by glycogen synthase (ca- kinase from human placental membrane phosphorylates porcine sein) kinase I [117], a member of a kinase group that operates brain tubulin and MAPs on tyrosine residues using [Y-’~P]ATP independent of activators such as CAMP and prefers acidic sub- as donor in a reaction enhanced 4-10-fold by insulin [125]. The strates. Casein kinase I incorporates 4 mol 32P/mol of tubulin u-subunit is modified about five-times more effectively than /?dimer, whereas CAMP-dependent kinase adds only 0.9 mol. p- tubulin. The results suggest that tubulin is a substrate for insulin tubulin is favoured over a-tubulin by both kinases. The tubulin receptor kinase in vivo and that phosphorylation upon exposure used in these experiments is from calf brain, but the kinase is to insulin or other hormones mediates changes in cellular microfrom rabbit skeletal muscle and not an endogenous brain enzyme tubules [1251. However, Eshraghi and Gotlieb [ 1261 demon[1171. Neural tubulin, if dephosphorylated, is modified in vitro strated in opposition to this proposal that insulin affects neither by casein kinase I1 from rat liver, mainly on a carboxy-terminal microtubule distribution within endothelial cells nor the reendoserine of a single P-subunit isoform [118]. However, when the thelialization of wounds. Purified epidermal-growth-factor(EGF)tubulin is not treated with phosphatase, serine and threonine resi- receptor kinase and Rous sarcoma virus src kinase also phosphodues are almost equally phosphorylated. Of importance when rylate microtubule proteins, respectively favouring MAP2 and assessing the role of casein kinase is that it is found in brain tubulin [127]. The EGF-receptor kinase, a protein similar to the and that it phosphorylates MAPZassociated CAMP-dependent insulin-receptor kinase, phosphorylates p-tubulin more readily kinase on its RII subunit. The kinase of neural origin targets than a, while the src kinase uses both subunits equally.

MacRae (Eur J. Biochem. 244)

27 1

Tyrosine kinases of 64 kDa and 46 kDa were partially puri- and occurs in the absence of a-tubulin phosphorylation. Phosfied from the membranes of Ehrlich ascites tumor and mouse phatase treatments reduce the amount of P: and increase fl indiliver cells [128]. The enzymes phosphorylate bovine brain tu- cating the relationship between these isoforms. The p2isoform, bulin, preferring a-tubulin to B-tubulin, in reactions independent thought to correspond to p2 (Gard and Kirschner, [133]), is type of EGF and insulin. A noteworthy characteristic of the two pro- 111p-tubulin and the phosphorylation was first reported to affect teins is that they share properties with kinase receptors of onco- either Ser444 or Ser446 via action of casein kinase I1 [119, 1351. gene products and growth promoters, implicating tubulin phos- Sequencing of a peptide from 3ZP-labelledrat brain tubulin rephorylation in cell proliferation. In this context, the mos proto- vealed that Ser444 of type I11 B-tubulin is the residue of interest oncogene product, pp39""', either associates with microtubules [136], a result confirmed by mass spectrometry and characterizawhen tubulin polymerization is favoured, or is found in a 500- tion of peptides obtained by CNBr cleavage of type 111p-tubulin kDa complex upon inhibition of assembly [129]. The ~ ~ 3 9 " " " from bovine brain [137]. Chemical analysis has verified that binds better to B-, than to a-tubulin, and the former is phosphor- type I11 p-tubulin is the isoform phosphorylated in brain and ylated more readily than the latter. The kinase localizes with indicates that residues such as Tyr437 and Ser259 may be modimetaphase spindles, and with asters and midbodies during telo- fied [138]. phase, but its action on microtubules within these structures is Appearance of phosphorylated serine during neuroblastoma uncertain. As another example of tyrosine kinase activity, a-tu- cell differentiation is indicative of p-tubulin modification during bulin and p-tubulin are modified during monocytic differentia- early development of mouse brain [119, 1331, a conclusion tion of HL-60 cells exposed in culture to tetradecanoyl phorbol strengthened by observation of in cultured mouse brain neuacetate [ 1301 and if phosphorylation is prevented so too is differ- rons [139] and in adult rats injected intracranially with entiation. Tyrosine kinases, members of the src-family of pro- ['*P]phosphate [136]. Sequence analysis suggests that type Ill pteins termed Fyn and Lyn, are induced as the cells differentiate tubulin interacts less well with MAPs than do other p-isoforms and a small portion of the enzymes may bind to intracellular and that phosphorylation governs its assembly [119, 1351. tubulin. It was proposed that phosphorylation of tubulin by Fyn Agreeing with this, stimulation of type I11 /I-tubulin assembly and Lyn alters tubulin assembly and thus monocyte development by MAP2 is lowered when phosphate is removed but this has [130]. no effect on polymerization in the absence of MAPs [138]. Jurkat T cells constitutively possess a low level of soluble Furthermore, the correlation between appearance of type I11 Ba-tubulin phosphorylated on tyrosine, and when they are stim- tubulin, its phosphorylation and the elaboration of neurites ulated via the T-cell receptor the amount of this tubulin increases would be characteristic of a mechanism whereby production of by 45% [131]. These results were obtained in experiments con- cell extensions depends on tubulin assembly in a process regutrolled for in vitro phosphorylation by chelating divalent cations lated by phosphorylation. In this context, the a-isotubulin is with EDTA and they suggest that the assembly of a tubulin sub- found within rat sciatic nerves where it may be restricted to population is regulated in activated T cells by tyrosine modifica- stable microtubules and confer discrete properties on these polytion. Although the enzyme was not identified in the work just mers [140]. described, T lymphocytes possess two protein kinases, ~ 5 6 ' and '~ Neurites are characterized by growth cones at their distal ~ 5 9 ~ ybelonging ", to the src family, whose members utilize tu- tips, cytoskeleton-enriched structures responsible for directing bulin [131]. However, because such a small fraction of tubulin growth and establishing contacts with synaptic targets [ 1411. is phosphorylated on tyrosine in Jurkat T cells, the physiological Within growth cones, tyrosine residues are phosphorylated by significance of the change is uncertain. Perhaps only microtu- pp6OC the prototype member of the src proto-oncogene fambule ends or a few tubulin isoforms are affected. Two tyrosine ily of non-receptor tyrosine kinases located at plasma-membrane kinases of Jurkat T cells, ZAP-70 and Vav, the latter a proto- inner surfaces. These kinases are signal-transducing enzymes, oncoprotein, bind constitutively to tubulin in a manner indepen- mediating differentiation and proliferation of cells. The major dent of one another [132]. Furthermore, in stimulated Jurkat T substrates of pp60c-s" in growth cones, revealed through use of cells there is preferential phosphorylation of tubulin-associated aniti-phosphotyrosine antibodies and "P-labelling, are populaZAP-70, raising the possibility that kinases are directed to ap- tions of membrane-associated tubulin [ 1411. Incorporation into propriate cellular locations through interaction with microtu- tyrosine residues, demonstrated in vivo for the first time in the bules. Thus microtubules may have a role in intracellular signal- work just mentioned, is 0.068 mol and 0.045 mol phosphate/mol ling and the potential for similar regulation of microtubule func- of a-tubulin and B-tubulin, respectively. Previously, in vivo phostion by other src-family kinases is illuminated. phorylation of tubulin was shown for serine residues [133], but tyrosine modification may have been missed because cell lysates Physiological relevance of tubulin phosphorylation. Phos- were prepared in the absence of sodium orthovanodate, an inhibphorylation of a single p-tubulin isoform, termed p2, increases itor of tyrosine phosphatases [1411. Phosphorylation of tubulin fourfold when neurite outgrowth is induced in N115 mouse neu- in vitro with pp60C-'" also occurs on a limited number of sites, roblastoma cells by serum withdrawal [ 1331. Precautions were and on peptides similar to those in vivo, with the exclusion of taken in this work to prevent artifactual 3zP incorporation into carboxy-terminal tyrosine. Matten et al. [ 1411 propose that microtubule proteins when they are purified by assembly/disas- pp60C-srcregulates microtubule dynamics, a conclusion reinsembly. Phosphorylation of B-tubulin, probably on a single ser- forced by inhibition of membrane tubulin phosphorylation in feine, is enhanced in both differentiated and undifferentiated N115 tal rat brain nerve growth cones upon activation of neural-cellcells by taxol-induced promotion of microtubule formation. adhesion molecules (N-CAMS) [142]. The NCAMs may relay Conversely, disassembly of microtubules upon exposure of cells signals into cells through their interaction with the cytoskeleton, to either nocodazole or colcemid reduces tubulin phosphoryla- and tubulin modification by enzymes such as pp60c-s'c is potention. It was proposed that the extent of phosphorylation mirrors tially important in this mechanism. The study of tubulin kinases has focused on neural systems the regulation of tubulin assembly during development and not the amount of kinase activity [133]. EddC et al. [134] demon- because they have an abundance of tubulin. Phosphorylated tustrated the post-translational phosphorylation in cultured neuro- bulin has, however, been seen in many tissues, some already blastomal cells of fi, a neuronal-specific p-tubulin. Appearance described. Other examples include uterine smooth muscle of rats correlates with neurite outgrowth where an estrogen-induced switching of a-tubulin and B-tubulin of the new isoform, called 6, ,'I'

272

MacRse (EUKJ. Biochem. 244)

phosphorylation is observed [ 1431. Tetrahymena cilia contain this modification [99, 1441, and about 10% of P-tubulin in the marginal bands of avian erythrocytes is phosphorylated at Ser441 [91]. The erythrocyte is interesting because it has a limited repertoire of tubulins, containing just a1 and p6 isoforms subject to only detyrosination and phosphorylation. However, the marginal bands of thrombin-activated and resting human platelets are said to lack phosphorylated tubulin [145], although there are reports to the contrary [146-1481. Overall, though, the cell types shown to have phosphorylated tubulin continue to increase, especially due to the work on tyrosine kinases, but the physiological relevance of this modification remains obscure. Phosphorylation may affect tubulin by direct perturbation of a reactive domain. Or, attachment of phosphate to a region causing a conformational change in tubulin could influence a distant site. Such changes potentially regulate binding of tubulin and microtubules to other cell components, as well as tubulin assembly and localization of microtubules to specific regions within cells. The latter is important because spatial organization is a key to microtubule function. A clear understanding of the role phosphorylation plays requires that those kinases whose actions in vivo have physiological consequences be identified with certainty and then characterized thoroughly. Moreover, study of tubulin dephosphorylation, limited in scope at the present time [115, 118, 134, 138, 149, 1501 is required.

Polyglutamylated tubulin Attachment of glutamyl units to carboxy-terminal glutamic acid residues of tubulin constitutes polyglutamylation. The modification was found in position 445 of Ma1 and Ma2 tubulin gene products by incubating mouse neurons in cycloheximide and ['Hlacetate, digesting the radioactive tubulin and sequencing peptides obtained by reverse-phase chromatography [151]. Mass spectrometry suggested that a glutamyl group, labelled by ['HIacetate during metabolism, is added to tubulin, a conclusion strengthened through labelling of tubulin upon incubation of cells with ['Hlglutamate. Glutamylation is thought to encompass 40-50% of a-tubulin in brain in a region of the protein that interacts with MAPS and Ca2+,thereby modulating tubulin assembly and microtubule organization [151]. Using a similar approach, Glu438 of bovine brain class I11 P-tubulin was shown to be glutamylated [152]. Up to 85% of this /I-tubulin isotype is affected and of this, at least 10% is phosphorylated at Ser444. Analysis of peptides obtained by limited subtilisin digestion demonstrates that pig brain tubulin is glutamylated at Glu435 and Glu445 of the &subunit and a-subunit, respectively [153, 1541. It seems that only a single glutamic acid residue on each tubulin polypeptide is targeted [154, 1551, and addition of the first glutamyl unit of the side chain is irreversible [156]. A major step in the study of polyglutamylation was production of the monoclonal antibody GT335, raised against a peptide corresponding to Glu441-Glu448 of Ma1 and Ma2 tubulin modified by addition of a yla2 biglutamylated peptide at Glu445 [157]. The epitope recognized by GT335 consists of the monoglutamylation motif and components of the nearby polypeptide. Probing of western blots with GT335 confirms glutamylation of both a-subunits and P-subunits of neuronal tubulin, while revealing that only P-tubulin is glutamylated in non-neural tissues, and this at very low levels. The observations suggest that one glutamylation enzyme is found in neural tissue and it utilizes a-tubulin, while a second enzyme recognizes P-tubulin and resides in many tissues, but at smaller amounts than in brain. In other work with GT335, glutamylated tubulin was found in 7: vaginalis [68], mouse sperm [158] and cultured melanophores from cod [159], indicating a broad distribution of this modification.

Absence of the glutamylated isoform in turkey erythrocytes, characterized by a marginal band of stable microtubules greatly enriched in detyrosinated tubulin, demonstrates that some cells lack the glutamylation enzyme(s) [91]. An important aspect in the study of glutamylation/deglutamylation enzymes is to characterize the chemical bonds between glutamic acids of the side chain. Clearly, the first residue is attached to the y-carboxyl group of a specific glutamic acid within tubulin [152], and more distal units must be linked to either acarboxyl or y-carboxyl groups by amide bonds to the preceding glutamate. By example, tetrahydrofolate is enzymatically glutamylated in Escherichia coli, with Glu2 and Glu3 adhering to the y-carboxyl group of the previous residue, while Glu4-8 bind to the a-carboxyl moiety [160, 1611. For mouse neural tubulin isotypes Ma1 and Ma2 the y-carboxyl group is the site for addition of the first glutamyl unit and the next two residues bind through their a-carboxyl groups [162]. The presence of two configurations suggests two enzymes; one adds the initial glutamate by a y-linkage and the second elongates the chain of a-carboxyl bonds. Conversely, one enzyme with two functions could be at work. In E. coli, two enzymes catalyze the formation of y- and a-bonds during glutamylation of tetrahydrofolate, but one of these may be able to mediate both reactions [161]. Additional study has demonstrated more than one y-linkage within the polyglutamylation modifications of tubulin. This enhances the complexity over that possible when variation is limited to the number of glutamyl residues and increases the likelihood that more than one enzyme is required for polyglutamylation of tubulin [163]. As significant as linkage mechanisms in determining function is the preference of glutamylating enzyme(s) for polymerized versus soluble tubulin. Detyrosinated neural tubulin is glutamylated, suggesting an association between the latter and microtubule stability [151]. However, about 80% of the tyrosinated isoform, itself approximately 13 % of total brain tubulin, is glutamylated. Because polyglutamylation affects a major portion of neuronal tyrosinated tubulin, an isoform usually unassembled or in labile microtubules, soluble tubulin also appears to be an enzyme substrate. The extent of glutamylation has no effect on a-tubulin detyrosination/tyrosination [ 1561, modifications dependent on the assembly state of tubulin, but the influence of detyrosination/tyrosination on glutamylation has yet to be determined. Moving from neuronal tissue, glutamylated tubulin occurs in stable and labile microtubules of Paramecium [ 1641. New microtubules in Paramecium are glutamylated, perhaps very soon after formation. However, the data, consisting of simultaneous microtubule recognition by GT335 and a general anti-tubulin antibody after assembly, allow for the possibility that microtubules arise from polyglutamylated tubulin. Incubation of young mouse neurons with ['H]glutamate, in concert with the use of GT335, revealed the reversible nature of tubulin polyglutamylation [ 1651. Moreover, glutamylation is inhibited when microtubules are disrupted by nocodozale, whereas stabilization with taxol either has no effect on the modification of a-tubulin or it stimulates glutamylation of the &subunit. Apparently, the glutamylation enzyme(s) prefers polymerized tubulin and the enzyme(s), which modifies a-tubulin as opposed to P-tubulin, is in limiting amounts. Deglutamylation of a-tubulin is insensitive to drugs, suggesting the responsible enzyme utilizes either polymerized or soluble tubulin, and the removal is biphasic with the outer glutamyl residues eliminated more rapidly than units 1-3 [165]. The a-tubulins with modifications 4-6 glutamyl residues long tend to be in microtubules while those with a smaller number of residues are distributed equally between polymer and dimer. Further, glutamylated ptubulin decreases markedly when cells are exposed to nocodo-

MacRae (Eul: J. Biochern. 244)

zale indicating that soluble P-tubulin is the preferred substrate for deglutamylation. The results just described support the requirement for several enzymes in the reversible glutamylation of a-tubulin and P-tubulin. Examination of isotubulin sequences and polyglutamylation patterns may reveal the number of glutamylation enzyme(s) and their recognition domains. Mouse testis isotubulins class I and IVb have different carboxy-terminal sequences but both are modified, implying multiple glutamylating enzymes each interacting with sites of different structure [ 1661. Another study, wherein glutamylation of mouse brain P-tubulin isotypes I and IVa are examined, favors the same conclusion [167]. The glutamylation enzyme(s) appeared not to recognize a common site, although it modifies a specific glutamyl residue in each isotubulin. Features shared by regions surrounding the glutamylated residue are an adjacent hydrophobic residue toward the amino end of the protein and an acidic residue two positions to the carboxy side. These properties are probably inadequate to dictate substrate specificity of the enzyme(s) [167]. Glutamylation of a-tubulin and P-tubulin during the growth of cultured neurons parallels glutamylation in developing brain [1551. In young neurons a-tubulin is extensively glutamylated with chains of 1-6 residues, and their complexity changes little over time. P-tubulin, however, is modified to a lesser degree in young neurons, but this gradually rises in the face of decreasing rates of glutamylation and deglutamylation. Moreover, incubation with nocodozale reduces the number of a5-a8 isoforms in the cytoskeleton but not in the soluble tubulin fraction, while the heterogeneity of P-tubulin glutamylation remains constant. Different enzymes may therefore glutamylate and deglutamylate a-tubulin and P-tubulin, with variations in catalytic rates and regulatory mechanisms [1551. Examination of developing mouse sperm, where isotypes ma3/7 and class IVb are glutamylated in sperm axonemes but not manchettes, indicates one way the responsible enzymes are regulated [166]. Perhaps enzymes are compartmentalized, lacking equal access to all tubulins. The appearance of glutamylated microtubules in the manchette of a mouse mutant, Olt/Olt, offers an experimental system for exploration of the compartmentalization proposal [166]. A model in which the extent of polyglutamylation determines tau binding by changing the conformation of tubulin suggests another enzyme regulatory mechanism [ 111. Three glutamyl units constitute the optimal length for binding of tau to microtubules and on either side of this number the MAP binds less efficiently. If the recognition site for the glutamylating enzyme includes both tubulin sequence and glutamylation units, then structural changes in tubulin promoted by side chains of different length may influence enzyme function. Rates of enzyme action could vary in this scheme, with polyglutamylation restricted to certain isotubulins and to specific glutamic acid residues in each isotype. The mechanism has the potential to work with one glutamylating enzyme or with several, and to also control deglutamylating enzymes.

Tubulin polyglycylation Polyglycylation is a post-translational modification of tubulin wherein 3 - 34 glycyl units are covalently attached to the y-carboxyl group of Glu445 in a-tubulin and Glu437 in P-tubulin [168]. Tubulin is the only protein known to be polyglycylated and the glycyl peptide is the longest known sequence of amino acids not genetically encoded. Immunological analysis, using the antibodies PAT and A X 0 49, shows that polyglycylation of tubulin is phylogenetically widespread [169]. About 60% of bull sperm P-tubulin possesses a glycyl chain up to 13 residues long attached to a glutamic acid residue by an isopeptide bond, but

273

a-tubulin is not modified [170]. Thus, in comparison to the Paramecium axoneme [ 1681, polyglycylation of bull sperm is less extensive. Levilliers et al. [171] found polyglycylated tubulin in quail and sea urchin axonemal tubulins, as well as in the cortex of Paramecium, with the distribution of the latter more restricted than acetylated a-tubulin. Polyglycylated tubulin is also present in the axonemes of Drosophila [I721 and sea urchin [67] sperm. Polyglycylated tubulin is detected in, and may be restricted to, very stable microtubules, perhaps appearing late in their formation [168, 171, 1721. Evidence that polyglycylation occurs on polymerized tubulin was obtained by examining the division of Paramecium. In this organism new microtubules are labelled with DMlA and DMlB, general monoclonal antibodies specific for epitopes at the carboxy terminus of a-tubulin and P-tubulin, respectively, before they react with antibodies to polyglycylated tubulin [ 1711. Furthermore, in Drosophila sperm, polyglycylated tubulin is first found during individualization, a time late in development when microtubules attach to the axonemal membrane [67, 1721. Interaction of microtubules with membranes may actually induce tubulin polyglycylation, an important possibility in Paramecium and Drosophila. Enzymes responsible for tubulin polyglycylation, or its reversal should this occur, have yet to be identified. Delayed glycylation of microtubules suggests that the enzyme prefers polymerized rather than soluble tubulin. Glycylation may involve successive modifications, with glycyl units added one at a time to microtubules, either by the same enzyme or a group of closely related enzymes [169, 1711. For example, two enzymes may be necessary for polyglycylation, one to attach the initial glycine to glutamic acid via a yCOOH-aNH, amide bond and a second to link glycine residues by aCOOH-aNH, bonds. Also, different enzymes, present in unequal amounts may be required to achieve variation in the labelling of a-tubulin and P-tubulin. As one possibility, glycyl dimers could combine with tubulin, as could preassembled oligomers which are trimmed after attachment. Whatever the case, a lag in the polyglycylation of stable microtubules in phylogenetically distant organisms supports the proposal that a similar enzymatic mechanism is employed by most cells [172]. Because transcription stops during elongation of Drosophila spermatids and before polyglycylation, this modification must be regulated at either translational or post-translational levels [ 1721. Possibly, the glycylation enzyme(s) is inactive during early sperm development, or not synthesized until it is required. The enzyme might be localized in the sperm head and distributed to the tail for interaction with tubulin only upon movement of the cystic bulge [172]. Or, the enzyme is sequestered along the axoneme, preventing its interaction with microtubules. Additionally, the glycylation enzyme(s) may associate with the cytoplasmic membrane and become active when microtubules and membranes meet [172]. A good way to answer this and related questions is to characterize the polyglycylation enzyme(s). Use of peptides to prepare the enzymes, in the same way that tubulin tyrosine-ligase was purified, is a viable approach [170]. The preference of the enzyme for different polymeric states of tubulin and for glycyl units of varying complexity could then be determined in vitro. Such data have the potential to reveal how the enzyme acts in vivo and to show how polyglycylation modulates microtubule function.

Conclusion Tubulin is subject to several different post-translational modifications, each carried out by a single enzyme or set of enzymes. Only one of these enzymes, namely tubulin tyrosineligase, has been purified to homogeneity and its gene cloned.

274

MacRae (Eur J. Biochem. 244)

Many of the remaining enzymes were characterized in cell-free extracts or after partial purification, risking interference by competing factors during their analysis. In other cases, properties were inferred by the study of modified tubulins within cells and the enzymes have yet to be studied. Potential exists, through enzyme studies, to realize important answers regarding tubulin post-translational modifications. For example, why some enzymes utilize polymerized tubulin while others target soluble tubulin is intriguing in terms of both enzyme mechanisms and tubulidmicrotubule function. Also of interest is whether post-translational modifications are restricted mostly to the carboxy terminus of the protein because this regulates MAP binding. For example, polyglutamylation and polyglycylation can yield bulky peptide side chains which shield reactive sites on the surface of microtubules and perturb the threedimensional conformation of tubulin, thereby influencing interaction with accessory proteins. Such modifications could ultimately determine the spatial distribution of microtubules and rapidly adjust their function without the need for synthesis of new proteins. Smaller changes, including the removal of tyrosine or glutamic acid, and the addition of acetyl and phosphate groups, may have more subtle consequences. The modifications may facilitate particular processes, such as tubulin assembly or generation of cell polarity, but not be absolutely required. However, because a selective advantage is accorded by virtue of the post-translational mechanisms, essential enzymes are maintained. It may therefore be misleading from the perspective of microtubule function to group the various tubulin modifications merely because they are posttranslational. Perhaps the most compelling reason to study these enzymes, in addition to elucidating mechanistic aspects of their activity, is that it may ultimately reveal the physiological relevance of tubulin post-translational modifications. The latter is a long standing issue and despite many eloquent experiments it has not been resolved for any tubulin modifications. Specifically, the purification of these enzymes will lead to cloning and sequencing of their genes. It then becomes possible, using the appropriate model organism or cultured cell, to either disrupt the gene or its mRNA product, effectively inhibiting enzyme synthesis. Conversely, the enzymes may be synthesized in cells or under conditions where their genes are not normally expressed. The effects on cells, ranging from minor functional disturbance to death could then be analyzed, indicating the role of tubulin post-translational modifications. Thus, opportunities to understand cytoskeleton function in eukaryotic cells through study of enzymes responsible for tubulin post-translational modifications are excellent.

REFERENCES 1. Fosket, D. E. & Morejohn, L. C. (1992) Structural and functional organization of tubulin, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 201-240. 2. Luduefia, R. F. (1993) Are tubulin isotypes functionally significant? Mol. Biol. Cell 4, 445-457. 3. Mitchison, T. J. (1992) Compare and contrast actin filaments and microtubules, Mol. Biol. Cell 3, 1309-1315. 4. Moudjou, M., Bordes, N., Paintrand, M. & Bornens, M. (1996) ytubulin in mammalian cells: the centrosomal and the cytosolic forms, J. Cell Sci. 109, 875-887. 5. Zheng, Y., Wong, M. L., Alberts, B. & Mitchison, T. (1995) Nucleation of microtubule assembly by a y-tubulin-containing ring complex, Nature 378, 578-583. 6 . Moritz, M., Braunfeid, M. B., Sedat, J. W., Alberts, B. & Agard, D. A. (1995) Microtubule nucleation by y-tubulin-containing rings in the centrosome, Nature 378, 638-640. 7. Lajoie-Mazenc, I., Tollon, Y., Detraves, C., Julian, M., Moisand, A., Gueth-Hallonet, C., Debec, A,, Salles-Passador, I., F'uget,

A,, Mazarguil, H., Raynaud-Messina, B. & Wright, M. (1994) Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle, J. Cell Sci. 107, 2825-2837. 8. MacRae, T. H. & Langdon, C. M. (1989) Tubulin synthesis, structure, and function: what are the relationships? Biochem. Celi Biol. 67, 770-790. 9. Cleveland, D. W. & Sullivan, K. F. (1985) Molecular biology and genetics of tubulin, Annu. Rev. Biochem. 54, 331-365. 10. MacRae, T. H. (1992) Towards an understanding of microtubule function and cell organization: an overview, Biochem. Cell Biol. 70, 835-841. 11. Boucher, D., Larcher, J.-C., Gros, F. & Denoulet, P. (1994) Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein tau and tubulin, Biochemistry 33, 12471- 12477. 12. Alfa, C. E. & Hyams, J. S . (1991) Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of a-tubulin, Cell Motil. Cytoskeleton 18, 86-93. 13. Wehland, J. & Weber, K. (1987) Turnover of the carboxy-terminal tyrosine of a-tubulin and means of reaching elevated levels of detyrosination in living cells, J. Cell Sci. 88, 185-203. 14. Bulinski, J. C., Richards, J. E. & Piperno, G. (1988) Posttranslational modifications of a-tubulin : detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells, J. Cell Biol. 106, 1213-1220. 15. Brown, A., Li, Y., Slaughter, T. & Black, M. M. (1993) Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules, J. Cell Sci. 104, 339-352. 16. Baas, P. W. & Black, M. M. (1990) Individual microtubules in the axon consist of domains that differ in both composition and stability, J. Cell Biol. 111, 495-509. 17. Pratt, L. F,, Okamura, S. & Cleveland, D. W. (1987) A divergent testis-specific a-tubulin isotype that does not contain a coded Cterminal tyrosine, Mol. Cell Biol. 7, 552-555. 18. Bulinski, J. C. & Gundersen, G. G. (1991) Stabilization and posttranslational modification of microtubules during cellular morphogenesis, Bioessays 13, 285-293. 19. Webster, D. R., Wehland, J., Weber, K. & Borisy, G. G. (1990) Detyrosination of alpha tubulin does not stabilize microtubules in vivo, J. Cell Biol. 111, 113-122. 20. Khawaja, S., Gundersen, G. G. & Bulinski, J. C. (1988) Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level, J. Cell Biol. 106, 141149. 21. Gundersen, G. G. & Bulinski, J. C. (1986) Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin, EM J. Cell Bid. 42, 288-294. 22. Paturle-Lafanechkre, L., Manier, M., Trigault, N., Pirollet, F., Mazarrguil, H. & Job, D. (1994) Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies, J. Cell Sci. 107, 1529- 1543. 23. Alonso, A. del C., Arce, C. A. & Barra, H. S. (1993) Tyrosinatable and non-tyrosinatable tubulin subpopulations in rat muscle in comparison with those in brain, Biochim. Biophys. Acta 1163, 26-30. 24. Gundersen, G. G., Khawaja, S. & Bulinski, J. C. (1987) Postpolymerization detyrosination of a-tubulin : a mechanism for subcelMar differentiation of microtubules, J. Cell Biol. 105, 251 -264. 25. Kreis, T. E. (1987) Microtubules containing detyrosinated tubulin are less dynamic, EMBO J. 6 , 2597-2606. 26. Webster, D. R., Gundersen, G. G., Bulinski, J. C. & Borisy, G. G. (1987) Assembly and turnover of detyrosinated tubulin in vivo, J. Cell Bid. 105, 265-276. D., Pirollet, F., Watterson, D. M., Haiech, J., 27. Bosc, C., Cronk, .I. Job, D. & Margolis, R. L. (1996) Cloning, expression, and properties of the microtubule-stabilizing protein STOP, Proc. Natl Acad. Sci. USA 93, 2125-2230. 28. Modesti, N. M., Argarafia, C. E., Barra, H. S . & Caputto, R. (1984) Inhibition of brain tubulinyl-tyrosine carboxypeptidase by endogenous proteins, J. Neurosci. Res. 12, 583-593.

MacRae (Eur J. Biochem. 244) 29. Argarafia, C. E., Barra, H. S . & Caputto, R. (1980) Tubulinyltyrosine carboxypeptidase from chicken brain : properties and partial purification, J. Neurochem. 34, 114- 118. 30. Argarafia, C. E., Barra, H. S . & Caputto, R. (1978) Release of ['4C]tyrosine from t~bulinyl-['~C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase, Mol. Cell. Biochem. 19, 17-21. 31. Kumar, N. & Flavin, M. (1981) Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin, J. Bid. Chem. 256, 7678-7686. 32. Alonso, A. del C., Arce, C. A. & Barra, H. S . (1988) Relationship between the tyrosination state of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development, Eus J. Biochem. 177, 517-522. 33. Beltramo, D. M., Arce, C. A. & Barra, H. S . (1989) Tyrosinationdetyrosination of tubulin and microtubules during the development of chick erythrocytes, Mol. Cell. Biochem. 89, 41-56. 34. Arce, C. A., Hallak, M. E., Rodriguez, J. A,, Barra, H. S . & Caputto, R. (1978) Capability of tubulin and microtubules to incorporate and to release tyrosine and phenylalanine and the effect of the incorporation of these amino acids on tubulin assembly, J. Neurochem. 31, 205-210. 35. Hallak, M. E., Rodriguez, J. A,, Barra, H. S . & Caputto, R. (1977) Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin, FEBS Lett. 73, 147-150. 36. Thompson, W. C. (1977) Post-translational addition of tyrosine to alpha tubulin in vivo in intact brain and in myogenic cells in culture, FEBS Lett. 80, 9-13. 37. Arce, C. A. & Barra, H. S . (1985) Release of C-terminal tyrosine from tubulin and microtubules at steady state, Biochem. J. 226, 3 1 1 -317. 38. Webster, D. R., Modesti, N. M. & Bulinski, J. C. (1992) Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubulebased assay, Biochemistry 31, 5849-5856. 39. Webster, D. R. & Oxford, M. G. (1996) Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydry-modifying compounds, J. Cell. Biochem. 60, 424-436. 40. Lopez, R. A., Arce, C. A. & Barra, H. S . (1990) Effect of polyanions and polycations on detyrosination of tubulin and microtubules at steady state, Biochim. Biophys. Acta 1039, 209-217. 41. Xiang, H. & MacRae, T. H. (1995) Production and utilization of detyrosinated tubulin in developing Artemia larvae: evidence for a tubulin-reactive carboxypeptidase, Biochem. Cell Biol. 73, 673 -685. 42. Arce, C. A. & Barra, H. S . (1983) Association of tubulinyl-tyrosine carboxypeptidase with microtubules, FEBS Lett. 157, 75 -78. 43. Weizetfel, J. C., Argarafia, C. E., Beltramo, D. M. & Barra, H. S . (1989) The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase, Biochem. Biophys. Res. Cornmun. 159, 770-776. 44. Deanin, G. G., Preston, S . F., Hanson, R. K. & Gordon, M. W. (1980) On the mechanism of turnover of the carboxy-terminal tyrosine of the alpha chain of tubulin, Eus J. Biochenz. 109, 201-216. 45. Barra, H. S . , Rodriguez, J. A,, Arce, C. A. & Caputto, R. (1973) A soluble preparation from rat brain that incorporates into its own proteins ['4C]arginine by a ribonuclease-sensitive system and ['4C]tyrosine by a ribonuclease-insensitive system, J. Neurochem. 20, 97-108. 46. Rodriguez, J. A,, Barra, H. S . , Arce, C. A,, Hallak, M. E. & Caputto, R. (1975) The reciprocal exclusion by L-dopa (~-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein, Biochem. J. 149, 115121. 47. Barra, H. S . , Arce, C. A., Rodriguez, J. A. & Caputto, R. (1973) Incorporation of phenylalanine as a single unit into rat brain protein : reciprocal inhibition by phenylalanine and tyrosine of their respective incorporations, J. Neurochem. 21, 1241- 1251. 48. Barra, H. S . , Arce, C. A,, Rodriguez, J. A. & Caputto, R. (1974) Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins, Biochem. Biophys. Res. Commun. 60, 1384-1390.

275

49. Arce, C. A., Rodriguez, J. A,, Barra, H. S . & Caputto, R. (1975) Incorporation of L-tyrosine, L-phenylalanine and ~-3,4-dihydroxyphenylalanine as single units into rat brain tubulin, EUK J. Biochem. 59, 145- 149. 50. Raybin, D. & Flavin, M. (1975) An enzyme tyrosylating a-tubulin and its role in microtubule assembly, Biochem. Biophys. Res. Commun. 65, 1088-1095. 51. Argaraiia, C. E., Arce, C. A,, Barra, H. S . & Caputto, R. (1977) In vivo incorporation of ['4C]tyrosine into the C-terminal position of the a subunit of tubulin, Arch. Biochem. Biophys. 180, 264268. 52. Raybin, D. & Flavin, M. (1977) Enzyme which specifically adds tyrosine to the a chain of tubulin, Biochemistry 16, 2189-2194. 53. Raybin, D. & Flavin, M. (1977) Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro, J. Cell Biol. 73, 492-504. 54. Preston, S . F., Deanin, G. G., Hanson, R. K. & Gordon, M. W. (1979) The phylogenetic distribution of tubulin: tyrosine ligase, J. Mol. E d . 13, 233-244. 55. Kobayashi, T. & Flavin, M. (1981) Tubulin tyrosylation in invertebrates, Conzp. Biochem. Physiol. B 69, 387-392. 56. Schroder, H. C., Wehland, J. & Weber, K. (1985) Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods, J. Cell Biol. 100, 276-281. 57. Murofushi, H. (1980) Purification and characterization of tubulintyrosine ligase from porcine brain, J. Biochem. (Tokyo) 87,979984. 58. Wehland, J. & Weber, K. (1987) Tubulin-tyrosine ligase has a binding site on P-tubulin: A two-domain structure of the enzyme, J. Cell Biol. 104, 1059-1067. 59. Beltramo, D. M., Arce, C. A. & Barra, H. S . (1987) Tubulin, but not microtubules, is the substrate for tubulin: tyrosine ligase in mature avian erythrocytes, J. Biol. Chem. 262, 15673- 15 677. 60. Rodriguez, J. A. & Borisy, G. G. (1979) Tyrosination state of free tubulin subunits and tubulin disassembled from microtubules of rat brain tissue, Biochem. Biophys. Res. Commuiz. 89, 893-899. 61. Riidiger, M., Wehland, J. & Weber, K. (1994) The carboxy-terminal peptide of detyrosinated a-tubulin provides a minimal system to study the substrate specificity of tubulin-tyrosine ligase, Eur: J. Biochem. 220, 309-320. 62. Ersfeld, K., Wehland, J., Plessmann, U., Dodemont, H., Volker, G. & Weber, K. (1993) Characterization of the tubulin-tyrosine ligase, J. Cell Biol. 120, 725-732. 63. Rodriguez, J. A. & Borisy, G. G. (1978) Modification of the Cterminus of brain tubulin during development, Biochem. Biophys. Res. Commun. 83, 579-586. 64. Barra, H. S., Arce, C. A. & Caputto, R. (1980) Total tubulin and its aminoacylated and non-aminoacylated forms during the development of rat brain, Eur: J. Biochem. 109, 439-446. 65. Paturle, L., Wehland, J., Margolis, R. L. & Job, D. (1989) Complete separation of tyrosinated, detyrosinated, and non-tyrosinatable brain tubulin subpopulations using affinity chromatography, Biochemistry 28, 2698-2704. 66. Paturle-Lafanecht-re, L., EddC, B., Denoulet, P., Van Dorsselaer, A,, Mazarguil, H., Le Caer, J. P., Wehland, J. & Job, D. (1991) Characterization of a major brain tubulin variant which cannot be tyrosinated, Biochemistry 30, 10523- 10528. 67. Mary, J., Redeker, V., Le Caer, J.-P., Rossier, J. & Schmitter, J.-M. (1996) Posttranslational modifications in the C-terminal tail of axonemal tubulin from sea urchin sperm, J. Biol. Chem. 271, 9928 -9933. 68. Delgado-Viscogliosi, P., Brugerolle, G. & Viscogliosi, E. (1996) Tubulin post-translational modifications in the primitive protist Trichomonas vaginalis, Cell Motil. Cytoskeleton 33, 288-297. 69. McKeithan, T. W. & Rosenbaum, J. L. (1981) Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of Polytomella, J. Cell Biol. 91, 352-360. 70. McKeithan, T. W., Lefebvre, P. A,, Silflow, C. D. & Rosenbaum, J. L. (1983) Multiple forms of tubulin in Polytomella and Chlamydomonas: Evidence for a precursor of flagellar a-tubulin, J. Cell Biol. 96, 1056-1063. 71. Lefebvre, P. A,, Silflow, C. D., Wieben, E. D. & Rosenbaum, J. L. (1980) Increased levels of mRNAs for tubulin and other flagellar

276

MacRae (Eut: J. Biochem. 244)

94. Gaertig, J., Cmz, M. A,, Bowen, J., Gu, L., Pennock, D. G. & proteins after amputation or shortening of Chlamydomonas flaGorovsky, M. A. (1995) Acetylation of lysine 40 in a-tubulin is gella, Cell 20, 469-477. not essential in Tetrakymena thennopkila, J. Cell Biol. 129, 72. L'Hernault, S. W. & Rosenbaum, J. L. (1983) Chlamydomonas (11301 -1310. tubulin is posttranslationally modified in the flagella during fla95. Goodman, D. B. P., Rasmussen, H., DiBella, F. & Guthrow, C. gellar assembly, J. Cell Bid. 97, 258-263. E. Jr (1970) Cyclic adenosine 3': 5'-monophosphate-stimulated 73. L'Hernault, S. W. & Rosenbaum, J. L. (1985) Reversal of the postphosphorylation of isolated neurotubule subunits, Proc. Nut1 translational modification on Cklamydomonas flagellar a-tubulin Acad. Sci. USA 67, 652-659. occurs during flagellar resorption, J. Cell Biol. 100, 457-462. 96. Eipper, B. A. (1972) Rat brain microtubule protein: purification 74. L'Hernault, S. W. & Rosenbaum, J. L. (1985) Cklumydomonas aand determination of covalently bound phosphate and carbohytubulin is posttranslationally modified by acetylation on the Edrate, Proc. Natl Acad. Sci. USA 69, 2283-2287. amino group of a lysine, Biochemistry 24, 473-478. 97. Eipper, B. A. (1974) Properties of rat brain tubulin, J. Biol. Chem. 75. LeDizet, M. & Piperno, G. (1987) Identification of an acetylation 249, 1407- 1416. site of Chlamydomonas a-tubulin, Proc. Nut1 Acad. Sci. USA 84, 98. Eipper, B. A. (1974) Rat brain tubulin and protein kinase activity, 5720-5724. J. Biol. Chem. 249, 1398-1406. 76. Brunke, K. J., Collis, P. S. & Weeks, D. P. (1982) Post-translational 99. Murofushi, H. (1973) Purification and characterization of a protein modification of tubulin dependent on organelle assembly, Nature 297, 516-518. kinase from Tetrahymena cilia, Biochim. Biopkys. Acta 327, 354-364. 77. Greer, K., Maruta, H., L'Hernault, S. W. & Rosenbaum, J. L. (1985) a-Tubulin acetylase activity in isolated Chlamydomonas 100. Piras, M. M. & Piras, R. (1974) Phosphorylation of vinblastineflagella, J. Cell Biol. 101, 2081-2084. isolated microtubules from chick-embryonic muscles, Eur J. 78. Sale, W. S., Besharse, J. C. & Piperno, G. (1988) Distribution of Biochem. 47,443-452. acetylated a-tubulin in retina and in in vitro-assembled microtu101. Shigekawa, B. L. & Olsen, R. W. (1975) Resolution of cyclic AMP bules, Cell Motil. Cytoskeleton 9, 243-253. stimulated protein kinase from polymerization-purified brain 79. Maruta, H., Greer, K. & Rosenbaum, J. L. (1986) The acetylation microtubules, Biochem. Biopkys. Res. Commun. 63, 455 -462. of alpha-tubulin and its relationship to the assembly and disas102. Sloboda, R. D., Rudolph, S. A,, Rosenbaum, J. L. & Greengard, P. sembly of microtubules, J. Cell Bid. 103, 571-579. (1975) Cyclic AMP-dependent endogenous phosphorylation of 80. Edd6, B., Rossier, J., Le Caer, J.-P., Berwald-Netter, Y., Koulakoff, a microtubule-associated protein, Proc. Natl Acad. Sci. USA 72, A,, Gros, E & Denoulet, P. (1991) A combination of post-trans177- 181. lational modifications is responsible for the production of neuro103. Reddington, M., Tan, L. P. & Lagnado, J. R. (1976) The phosphornal a-tubulin heterogeneity, J. Cell. Biochem. 46, 134- 142. ylation of brain microtubular proteins in situ and in vitro, J. 81. Lloyd, R. A., Gentleman, S. & Chader, G. J. (1994) Assay of tuNeurockem. 27, 1229-1236. bulin acetyltransferase activity in subcellular tissue fractions, 104. Forgue, S. T. & Dahl, J. L. (1979) Rat brain tubulin: subunit Anal. Biockem. 216, 42-46. heterogeneity and phosphorylation, J . Neurochem. 32, 101582. LeDizet, M. & Piperno, G. (1986) Cytoplasmic microtubules con1025. taining acetylated a-tubulin in Chlamydomonas reinhardtii: spa105. Burke, B. E. & DeLorenzo, R. J. (1981) Ca2+- and calmodulintial arrangement and properties, J. Cell Biol. 103, 13-22. stimulated endogenous phosphorylation of neurotubulin, Proc. 83. Piperno, G., LeDizet, M. & Chang, X.-J. (1987) Microtubules conNut1 Acad. Sci. USA 78, 991-995. taining acetylated a-tubulin in mammalian cells in culture, J. 106. Burke, B. E. & DeLorenzo, R. J. (1982) Ca2+ and calmodulinCell Biol. 104, 289-302. regulated endogenous tubulin kinase activity in presynaptic 84. Schatten, G., Simerly, C., Asai, D. J., Sz6ke, E., Cooke, P. & Schatnerve terminal preparations, Brain Res. 236, 393-415. ten, H. (1988) Acetylated a-tubulin in microtubules during 107. Burke, B. E. & DeLorenzo, R. J. (1982) Caz+ and calmodulinmouse fertilization and early development, Dew.Bid. 130, 74dependent phosphorylation of endogenous synaptic vesicle tu86. bulin by a vesicle-bound calmodulin kinase system, J. Neu85. Cambray-Deakin, M. A. & Burgoyne, R. D. (1987) Acetylated and rochem. 38, 1205-1218. detyrosinated a-tubulins are co-localized in stable microtubules 108. Goldenring, J. R., Gonzalez, B., McGuire, J. S. Jr & DeLorenzo, in rat meningeal fibroblasts, Cell Motil. Cytoskeleton 8, 284R. J. (1983) Purification and characterization of a calmodulin291. dependent kinase from rat brain cytosol able to phosphorylate 86. Wilson, P. J. & Forer, A. (1989) Acetylated a-tubulin in spermatotubulin and microtubule-associated proteins, J. Bid. Chem. 258, genic cells of the crane fly Nepkrotoma suturalis: kinetochore 12632- 12 640. microtubules are selectively acetylated, Cell Motil. Cytoskeleton 109. Goldenring, J. R., Casanova, J. E. & DeLorenzo, R. J. (1984) Tu14, 237-250. bulin-associated calmodulin-dependent lunase : evidence for an 87. Wolf, N., Regan, C. L. & Fuller, M. T. (1988) Temporal and spatial endogenous complex of tubulin with a calcium-calmodulin-depattern of differences in microtubule behaviour during Drosophpendent kinase, J. Neurockem. 43, 1669- 1679. ila embryogenesis revealed by distribution of a tubulin isoform, 110. Goldenring, J. R., McGuire, J. S. Jr & DeLorenzo, R. J. (1984) Development (Cambx) 102, 31 1-324. Identification of the major postsynaptic density protein as ho88. Chu, D. T. W. & Klymkowsky, M. W. (1989) The appearance of mologous with the major calmodulin-binding subunit of a calacetylated a-tubulin during early development and cellular difmodulin-dependent protein kinase, J. Neurochem. 42, 1077ferentiation in Xenopus, Dev. Biol. 136, 104-117. 1084. 89. Webster, D. R. & Borisy, G. G. (1989) Microtubules are acetylated 1 1 2 . Larson, R. E., Goldenring, J. R., Vallano, M. L. & DeLorenzo, in domains that turn over slowly, J. Cell Sci. 92, 57-65. R. J. (1985) Identification of endogenous calmodulin-dependent 90. Lim, S.-S., Sammak, P. J. & Borisy, G. G. (1989) Progressive and kinase and calmodulin-binding proteins in cold-stable microtuspatially differentiated stability of microtubules in developing bule preparations from rat brain, J. Neurockem. 44, 1566-1574. neuronal cells, J. Cell B i d . 109, 253-263. 112. Suwita, E., Lapadula, D. M. & Abou-Donia, M. B. (1986) Calcium 91. Rudiger, M. & Weber, K. (1993) Characterization of the post-transand calmodulin-enhanced in vitro phosphorylation of hen brain lational modifications in tubulin from the marginal baud of avian cold-stable microtubules and spinal cord neurofilament triplet erythrocytes, Eut: J . Biochem. 218, 107-1 16. proteins after a single oral dose of tri-o-cresyl phosphate, Proc. 92. Read, M., Sherwin, T., Holloway, P., Gull, K. & Hyde, J. E. (1993) Natl Acad. Sci. USA 83, 6174-6178. Microtubule organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falci- 113. Fukunaga, K., Yamamoto, H., Matsui, IS.,Higashi, K. & Miyamoto, E. (1982) Purification and characterization of a Ca2+-and parum and investigation of post-translational modifications of calmodulin-dependent protein kinase from rat brain, J. Neuroparasite tubulin, Parasitology 106, 223 -232. ckem. 39, 2607-1617. 93. Kozminski, K. G . , Diener, D. R. & Rosenbaum, J. L. (1993) High 114. Yamamoto, H., Fukunaga, K., Goto, S., Tanaka, E. & Miyamoto, level expression of nonacetylatable a-tubulin in Chlamydomonas E. (1985) Ca2+,calmodulin-dependent regulation of microtubule reinkardtii, Cell Motil. Cytoskeleton 25, 158- 170.

277

MacRae (Eur: J. Biochem. 244)

115. 116. 117.

118. 119. 120. 121.

122. 123. 124. 125.

126. 127.

128.

129.

130.

131.

132.

133.

134.

formation via phosphorylation of microtubule-associated protein 2, t factor, and tubulin, and comparison with the cyclic AMPdependent phosphorylation, J. Neurochem. 44, 759-768. Wandosell, F., Serrano, L., HernBndez, M. A. & Avila, J. (1986) Phosphorylation of tubulin by a calmodulin-dependent protein kinase, J. Biol. Chem. 261, 10332-10339. Hargreaves, A. J., Wandosell, F. & Avila, J. (1986) Phosphorylation of tubulin enhances its interaction with membranes, Nature 323, 827-828. Singh, T. J., Akatsuka, A,, Huang, K.-P., Murthy, A. S. N. & Flavin, M. (1984) Cyclic nucleotide- and Ca2'-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1, Biochem. Biophys. Res. Commun. 121, 19-26. Serrano, L., Diaz-Nido, J., Wandosell, F. & Avila, J. (1987) Tubulin phosphorylation by casein kinase I1 is similar to that found in vivo, J. Cell Biol. 105, 1731-1739. Avila, J., Serrano, L., Hernindez, M. A. & Diaz-Nido, J. (1988) Phosphorylation of neuronal microtubule proteins, Protoplasma 145, 82-88. Serrano, L., Hernindez, M. A., Diaz-Nido, J. & Avila, J. (1989) Association of casein kinase I1 with microtubules, Exp. Cell Res. 181, 263-272. Risnik, V. V., Adim, G., Gusev, N. B. & Friedrich, P. (1988) Casein kinases I and I1 bound to pig brain microtubules, Cell. Mol. Neurobiol. 8, 315-324. Crute, B. E. & Van Buskirk, R. G. (1992) A casein kinase-like kinase phosphorylates p-tubulin and may be a microtubule-associated protein, J. Neurochem. 59, 2017-2023. Takahashi, M., Tomizawa, K., Sato, K., Ohtake, A. & Omori, A. (1995) A novel tau-tubulin kinase from bovine brain, FEBS Lett. 372, 59-64. Wandosell, F., Serrano, L. & Avila, J. (1987) Phosphorylation of atubulin carboxy-terminal tyrosine prevents its incorporation into microtubules, J. Biol. Chem. 262, 8268-8273. Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S . , Akanuma, Y. & Kasuga, M. (1985) Phosphorylation of tubulin and microtubule-associated proteins by the purified insulin receptor kinase, J . Biol. Chem. 260, 40164020. Eshraghi, S. & Gotlieb, A. I. (1995) Insulin does not disrupt actin microfilaments, microtubules, and in vitro aortic endothelial wound repair, Biochem. Cell Biol. 73, 507-514. Akiyama, T., Kadowaki, T., Nishida, E., Kadooka, T., Ogawara, H., Fukami, Y., Sakai, H., Takaku, F. & Kasuga, M. (1986) Substrate specificities of tyrosine-specific protein kinases toward cytoskeleta1 proteins in vitro, J. Biol. Chem. 261, 14797-14803. Yoshikawa, K., Usui, H., Imazu, M., Tuskamoto, H. & Takeda, M. (1985) Comparison of tyrosine protein kinases in membrane fractions from mouse liver and Ehrlich ascites tumor, J. Biol. Chem. 260, 15091-15099. Zhou, R., Oskarsson, M., Paules, R. S., Schulz, N., Cleveland, D. & Vande Woude, G. F. (1991) Ability of the c-mos product to associate with and phosphorylate tubulin, Science 251, 671 675. Katagiri, K., Katagiri, T., Kajiyama, K., Yamamoto, T. & Yoshida, T. (1993) Tyrosine-phosphorylation of tubulin during monocytic differentiation of HL-60 cells, J. Immunol. 150, 585-593. Ley, S. C., Verbi, W., Pappin, D. J. C., Druker, B., Davies, A. A. & Crumpton, M. J. (1994) Tyrosine phosphorylation of a-tubulin in human T lymphocytes, Eur: J. lmmunol. 24, 99-106. Huby, R. D. J., Carlile, G. W. & Ley, S . C. (1995) Interactions between the protein- tyrosine kinase ZAP-70, the proto-oncoprotein Vav, and tubulin in Jurkat T cells, J. Biol. Chem. 270, 30241 -30244. Gard, D. L. & Kirschner, M. W. (1985) A polymer-dependent increase in phosphorylation of p-tubulin accompanies differentiation of a mouse neuroblastoma cell line, J. Cell Biol. 100,764774. Eddt, B., De Nechaud, B., Denoulet, P. & Gros, F. (1987) Control of isotubulin expression during neuronal differentiation of mouse neuroblastoma and teratocarcinoma cell lines, Dev. Biol. 123, 549-558.

135. Ludueiia, R. F., Zimmerniann, H.-P. & Little, M. (1988) Identification of the phosphorylated p-tubulin isotype in differentiated neuroblastoma cells, FEBS k t t . 230, 142-146. 136. Diaz-Nido, J . , Serrano, L., Lopez-Otin, C., Vandekerckhove, J. & Avila, J. (1990) Phosphorylation of a neuronal-specific P-tubulin isotype, J. Biol. Chem. 265, 13949-13954. 137. Alexander, J. E., Hunt, D. F., Lee, M. K., Shabanowitz, J., Michel, H., Berlin, S. C . , MacDonald, T. L.. Sundberg, R. J., Rebhun, L. I. & Frankfurter, A. (1991) Characterization of post-translational modifications in neuron-specific class 111 P-tubulin by mass spectrometry, Proc. Natl Acad. Sci. USA 88, 4685-4689. 138. Khan, I. A. & Ludueiia, R. F. (1996) Phosphorylation of PII-tubulin, Biochemistry 35, 3704-3711. 139. EddC, B., Denoulet, P., de Nechaud, B., Koulakoff, A,, BerwaldNetter, Y. & Gros, F. (1989) Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia, Biol. Cell. 65, 109-117. 140. Denoulet, P., Filliatreau, G., de Nkchaud, B., Gros, F. & Di Giamberardino, L. (1989) Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve, J. Cell Biol. 108, 965 -971. 141. Matten, W. T., Aubry, M., West, J. & Maness, P. F. (1990) Tubulin is phosphorylated at tyrosine by pp60c-s" in nerve growth cone membranes, J. Cell Biol. 111, 1959-1970. 142. Atashi, J. R., Klinz, S. G., Ingraham, C. A,, Matten, W. T., Schachner, M. & Maness, P. F. (1992) Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes, Neuron 8, 831-842. 143. Joseph, M. K., Fernstrom, M. A. & Soloff, M. S. (1982) Switching of p- to a-tubulin phosphorylation in uterine smooth muscle of parturient rats, J . Biol. Chem. 257, 11728-11733. 144. Hirano-Ohnishi, J. & Watanabe, Y. (1989) Ca*'/calmodulin-dependent phosphorylation of ciliary P-tubulin in Tetrahymena, J. Biochem. (Tokyo) 105, 858-860. 145. Janiak, A., Villar, R., Cassoly, R. & Rendu, F. (1995) Tubulin is not phosphorylated in resting and thrombin-activated platelets, J. Biochem. (Tokyo) 117, 296-302. 146. Berry, S., Dawicki, D. D. & Steiner, M. (1989) Time resolved analysis of tubulin phosphorylation during platelet activation, Biochem. Biophys. Res. Commun. 159, 170- 176. 147. Berry, S., Dawicki, D. & Steiner, M. (1988) Agonist-induced changes of platelet tubulin phosphorylation, Biochem. Biophys. Res. Commun. 151, 1250-1255. 148. Ikeda, Y. & Steiner, M. (1979) Phosphorylation and protein kinase activity of platelet tubulin, J. Biol. Chem. 254, 66-74. 149. Li, S. & Donelson, J. E. (1995) Inhibition of protein phosphatase 1 and 2A down-regulates 8-tubulin gene expression in Trypanosoma rhodesiense, Biochem. Biophys. Res. Commun. 212, 793 799. 150. Yamamoto, H., Saitoh, Y., Fukunaga, K., Nishimura, H. & Miyamoto, E. (1988) Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly, J. Neurochem. 50, 1614- 1623. 151. Eddt, B., Rossier, J., Le Caer, J.-P., Desbruyeres, E., Gros, F. & Denoulet, P. (1990) Posttranslational glutamylation of a-tubulin, Science 247, 83 - 85. 152. Alexander, J. E., Hunt, D. F., Lee, M. K., Shabanowitz, J., Michel, H., Berlin, S . C., MacDonald, T. L., Sundberg, R. I., Rebhun, L. I. & Frankfurter, A. (1991) Characterization of post-translational modifications in neuron-specific class I11 p-tubulin by mass spectrometry, Proc. Natl Acad. Sci. USA 88, 4685-4689. 153. Redeker, V., Melki, R., PromC, D., Le Caer, J.-P. & Rossier, J. (1992) Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major a and P tubulin isotypes from pig brain are glutamylated, FEBS Lett. 313, 185-192. 154. Riidiger, M., Plessman, U., Kloppel, K.-D., Wehland, J. & Weber, K. (1992) Class I1 tubulin, the major brain tubulin isotype is polyglutamylated on glutamic acid residue 435, FEBS Lett. 308, 101-105. 155. Audebert, S., Koulakoff, A,, Benvald-Netter, Y., Gros, F., Denoulet, P. & Eddt, B. (1994) Developmental regulation of polyglutamylated a- and p-tubulin in mouse brain neurons, J . Cell Sci. 107, 2313-2322.

P

278

MacRae (Euc J. Biochem. 244)

156. Eddt, B., Rossier, J., Le Caer, J.-P., PromC, J. C., Desbruykres, E., Gros, F. & Denoulet, P. (1992) Polyglutamylated a-tubulin can enter the tyrosinationldetyrosination cycle, Biochemistry 31, 403-410. 157. Wolff, A,, de Ntchaud, B., Chillet, D., Mazarguil, H., Desbruykres, E., Audebert, S., EddC, B., Gros, F. & Denoulet, P. (1992) Distribution of glutamylated a and P-tubulin in mouse tissues using a specific monoclonal antibody, GT335, Euc J. Cell Biol. 59, 425-432. 158. Fouquet, J . 2 , Prigent, Y. & Kann, M.-L. (1996) Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum : unique distribution of glutamylated tubulin, Mol. Reprod. Dev. 43, 358-365. 159. Nilsson, H., Rutberg, M. & Wallin, M. (1996) Localization of kinesin and cytoplasmic dynein in cultured melanophores from Atlantic cod, Gadus morhua, Cell Motil. Cytoskeleton 33, 183196. 160. Ferone, R., Hanlon, M. H., Singer, S . C. & Hunt, D. F. (1986) aCarboxyl-linked glutamates in the folypolyglutamates of Escherichia coli, J. Biol. Chem. 261, 16356-16362. 161. Ferone, R., Singer, S. C. & Hunt, D. F. (1986) In vitro synthesis of a-carboxyl-linked folypolyglutamates by an enzyme preparation from Escherichia coli, J. Biol. Chem. 261, 16363-16371. 162. Redeker, V., Le Caer, J.-P., Rossier, J. & PromC, J.-C. (1991) Structure of the polyglutamyl side chain posttranslationally added to a-tubulin, 1. Biol. Chem. 266, 23461-23466. 163. Wolff, A., Houdayer, M., Chillet, D., de Ntchaud, B. & Denoulet, P. (1994) Structure of the polyglutamyl chain of tubulin: occurrence of alpha and gamma linkages between glutamyl units revealed by monoreactive polyclonal antibodies, Biol. Cell 81, 11-16. 164. BrC, M. H., de NCchaud, B., Wolff, A. & Fleury, A. (1994) Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335, Cell Motil. Cytoskeleton 27, 337 -349.

165. Audebert, S., Desbruyitres, E., Gruszczynski, C., Koulakoff, A, Gros, F., Denoulet, P. & EddC, B. (1993) Reversible polygluramylation of a- and P-tubulin and microtubule dynamics in mouse brain neurons, Mol. Biol. Cell 4, 615-626. 166. Fouquet, J.-P., EddC, B., Kann, M.-L., Wolff, A., Desbruykres, E. & Denoulet, P. (1994) Differential distribution of glutamylated tubulin during spermatogenesis in mammalian testis, Cell Moi'il. Cytoskeleton 27, 49 -5 8. 167. Mary, J., Redeker, V., Le Caer, J.-P., Prome, J.-C. & Rossier, J. (1994) Class I and IVa P-tubulin isotypes expressed in adutt mouse brain are glutamylated, FEBS Lett. 353, 89-94. 168. Redeker, V., Levilliers, N., Schmitter, J.-M., LeCaer, J.-P., Rossilx, J., Adoutte, A. & Bre, M.-H. (1994) Polyglycylation of tubulin: a post-translational modification in axonemal microtubules, Strience 266, 1688-1691. 169. BrC, M.-H., Redeker, V., Quibell, M., Darmanaden-Delorme, J., Bressac, C., Cosson, J., Huitorel, P.. Schmitter, J.-M., Rossier, J., Johnson, T., Adoutte, A. & Levilliers, N. (1996) Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility, J. Cell Sci. 109, 727-738. 170. Rudiger, M., Plessmann, U., Rudiger, A.-H. & Weber, K. (1995) P tubulin of bull sperm is polyglycylated, FEBS Lett. 364, 147151. 171. Levilliers, N., Fleury, A. & Hill, A.-M. (1995) Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin, J. Cell Sci. 108, 3013-3028. 172. Bressac, C., BrC, M.-H., Darmanaden-Delorme, J., Laurent, hi., Levilliers, N. & Fleury, A. (1995) A massive new post-translational modification occurs on axonemal tubulin at the final stc:p of spermatogenesis in Drosophila, Euc J. Cell Biol. 67, 346355.